Covalently linked oligonucleotide minor groove binder conjugates

ABSTRACT

Minor groove binding molecules are covalently bound to oligonucleotides which in their base sequence are complementary to a target sequence of single stranded or double stranded DNA, RNA or hybrids thereof. The covalently bound oligonucleotide minor groove binder conjugates strogly bind to the target sequence of the complementary strand.

This application is a continuation of and claims the benefit of U.S.Pat. No. 09/507,345, filed Feb. 18, 2000, which is a continuation ofU.S. Pat. No. 09/141,764, filed Aug. 27, 1998—now U.S. Pat. No.6,084,102, which is a continuation of U.S. Pat. No. 08/415,370, filedApr. 3, 1995—now U.S. Pat. No. 5,801,155, the disclosure of each beingincorporated by reference.

FIELD OF THE INVENTION

The present invention is directed to new derivatives ofoligonucleotides. More particularly, the present invention is directedto oligonucleotide derivatives wherein one or more minor groove bindingmolecules are covalently attached to the oligonucleotide. Theoligonucleotide minor groove binding moiety conjugates show strongaffinity to hybridize and strongly bind to complementary sequences ofsingle or double stranded nucleic acids, and thereby have utility assequence specific probes and as antisense and anti-gene therapeuticagents.

BRIEF DESCRIPTION OF THE PRIOR ART

Minor groove binding agents which non-covalently bind into the minorgroove of double stranded DNA are known in the art. Intercalating agentswhich bind to double stranded DNA or RNA are also well known in the art.Intercalating agents are, generally speaking, flat aromatic moleculeswhich non-covalently bind to double stranded DNA or RNA by positioning(intercalating) themselves between interfacing purine and pyrimidinebases of the two strands of double stranded DNA or RNA. U.S. Pat. No.4,835,263 describes oligonucleotides which are covalently bound to anintercalating group. Such oligonucleotides carrying an intercalatinggroup can be useful as hybridization probes.

SUMMARY OF THE INVENTION

The present invention relates to a covalently bound oligonucleotide andminor groove binder combination which includes an oligonucleotide havinga plurality of nucleotide units, a 3′-end and a 5′-end, and a minorgroove binder moiety covalently attached to at least one of saidnucleotides. The minor groove binder is typically attached to theoligonucleotide through a linking group comprising a chain of no morethan 15 atoms. The minor groove binder moiety is a radical of a moleculehaving a molecular weight of approximately 150 to approximately 2000Daltons which molecule binds in a non-intercalating manner into theminor groove of double stranded DNA, RNA or hybrids thereof with anassociation constant greater than approximately 10³ M⁻¹.

In another aspect, the present invention relates to the process ofsynthesizing certain covalently bound oligonucleotide minor groovebinder combinations, and to the manner of using such combinations forhybridization probe and related analytical and diagnostic, as well astherapeutic (anti-sense and anti-gene) purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the results of a slot blot hybridizationassay. Sequences 1-4 SEQ ID NOS: 9-12.

DETAILED DESCRIPTION OF THE INVENTION GENERAL EMBODIMENTS

A prominent feature of the novel composition of matter of the presentinvention is that a minor groove binding molecule is covalently bound toan oligoneucleotide. As is noted in the introductory section of thepresent application for patent, a minor groove binder is a molecule thatbinds within the minor groove of double stranded deoxyribonucleic acid(DNA). Although a general chemical formula for all known minor groovebinding compounds cannot be provided because such compounds have widelyvarying chemical structures, compounds which are capable of binding inthe minor groove of DNA, generally speaking, have a crescent shape threedimensional structure. Most minor groove binding compounds of the priorart have a strong preference for A-T (adenine and thymine) rich regionsof the B form of double stranded DNA. The minor groove bindingcompounds, or more accurately stated moieties of theoligonucleotide-minor groove binding conjugates of the presentinvention, also have the same preference. (The oligonucleotide-minorgroove binding conjugates of the present invention are hereinaftersometimes referred to as ODN-MGB.) Nevertheless, minor groove bindingcompounds which would show preference to C-G (cytosine and guanine) richregions are also theoretically possible. Therefore, ODN-MGB compoundsincorporating a radical or moiety derived from minor groove bindermolecules having preference for C-G regions are also within the scope ofthe present invention. The preference for A-T regions of the known minorgroove binders is currently explained by the existence of an unfavorablesteric interference between the 2-amino group of guanine and some wellknown minor groove binders. However, as it will become apparent from theensuing further description, when guanine is replaced by hypoxanthine inan ODN-MGB of the present invention, the potential for the above-notedunfavorable steric interference no longer exists and strong binding ofthe ODN-MGB to a complementary strand may occur.

Generally speaking, minor groove binding compounds known in the priorart do not bind to double stranded RNA or to a double stranded hybrid ofDNA and RNA. However, the ODN-MGB compounds of the present inventionexhibit potential for binding to single stranded RNA, and the foregoingfeature forms another interesting and novel aspect of the presentinvention.

Examples of known minor groove binding compounds of the prior art, whichcan, in accordance with the present invention, be covalently bound toODNs to form the novel ODM-MGB conjugates are certain naturallyoccurring compounds such as netropsin, distamycin and lexitropsin,mithramycin, chromomycin A₃, olivomycin, anthramycin, sibiromycin, aswell as further related antibiotics and synthetic derivatives. Certainbisquarternary ammonium heterocyclic compounds, diarylamidines such aspentamidine, stilbamidine and berenil, CC-1065 and related pyrroloindoleand indole polypeptides, Hoechst 33258, 4′-6-diamidino-2-phenylindole(DAPI) as well as a number of oligopeptides consisting of naturallyoccurring or synthetic amino acids are minor groove binder compounds.The chemical structures of the following examples are illustrated below.

For the purposes of the present invention a molecule is a minor groovebinder if it is capable of binding within the minor groove of doublestranded DNA with an association constant of 10³ M⁻¹ or greater. Thistype of binding can be detected by well established spectrophotometricmethods, such as ultraviolet (u.v.) and nuclear magnetic resonance (nmr)spectroscopy and also by gel electrophoresis. Shifts in u.v. spectraupon binding of a minor groove binder molecule, and nmr spectroscopyutilizing the “Nuclear overhauser” (NOSEY) effect are particularly wellknown and useful techniques for this purpose. Gel electrophoresisdetects binding of a minor groove binder to double stranded DNA orfragment thereof, because upon such binding the mobility of the doublestranded DNA changes.

Intercalating molecules or agents are readily distinguished from minorgroove binders on the basis that the intercalating agents are flataromatic (preferably polycyclic) molecules versus the “crescent shape”or analogous geometry of the minor groove binders. An experimentaldistinction can also be made by nmr spectroscopy utilizing the NuclearOverhauser effect.

As noted above, for the purposes of the present invention a molecule isa minor groove binder if its association constant within the minorgroove of double stranded DNA is 10³ M⁻¹ or greater. However, some minorgroove binders bind to the high affinity sites of double stranded DNAwith an association constant of the magnitude of 10⁷ to 10⁹ M ⁻¹ Inaccordance with the present invention, the minor groove binder moleculeis derivatized, in essence formed into a “radical” and linked to anappropriate covalent structure or chain of atoms that attaches the minorgroove binder to the ODN. In a sense, the linking “chain” can andsometimes is considered as part of the minor groove binder since thenature of the linkage is such that it does not adversely affect theminor groove binding properties of the ODN-MGB molecule. However, itsuits the present description better to conceptually separate the minorgroove binder from the group that covalently attaches it to the ODN. Theradical “formed” from the minor group binder molecule is hereinafterreferred to as the “minor groove binder moiety”, and the covalentlinkage (which may be a chain of up to approximately 15 atoms) thatattaches the minor groove binder moiety to the oligonucleotide is calledthe “linking group”. The preferred embodiments of the minor groovemoieties in accordance with the present invention are described indetail after description of the oligonucleotide portion of the ODN-MGBconjugate compounds of the present invention.

Broadly speaking, the oligonucleotide portion of the ODN-MGB conjugatesof the present invention comprise approximately 3 to 100 nucleotideunits. The nucleotide units which can be incorporated into the ODNs inaccordance with the present invention include the major heterocyclicbases naturally found in nucleic acids (uracil, cytosine, thymine,adenine and guanine) as well as naturally occurring and syntheticmodifications and analogs of these bases such as hypoxanthine,2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-N⁴ ethenocytosine,4-aminopyrrazolo[3,4-d]pyrimidine and6-amino-4-hydroxy-[3,4-d]pyrimidine. The respective structures of the2-deoxyribosides of 5-N⁴ ethenocytosine4-aminopyrrazolo[3,4-d]pyrimidine and of6-amino-4-hydroxy-[3,4-d]pyrimidine are shown below.

In addition, the nucleotide units which are incorporated into the ODNsof the ODN-MGB conjugates of the present invention may have across-linking function (an alkylating agent) covalently bound to one ormore of the bases, through a linking arm. Since the ODN-MGB conjugateshaving an attached cross-linking agent form an important class ofpreferred embodiments of the present invention these structures will bedescribed in more detail below.

The “sugar” or glycoside portion of the ODN-MGBs of the presentinvention may comprise deoxyribose, ribose, 2-fluororibose, 2-O alkyl oralkenylribose where the alkyl group may have 1 to 6 carbons and thealkenyl group 2 to 6 carbons. In the naturally occurring nucleotides andin the herein described modifications and analogs the deoxyribose orribose moiety forms a furanose ring, the glycosydic linkage is of the μconfiguration and the purine bases are attached to the sugar moiety viathe 9-position, the pyrimidines via the 1-position and thepyrazolopyrimidines via the 1-position. Presently,oligodeoxyribonucleotides are preferred in accordance with the presentinvention, therefore the preferred sugar is 2-deoxyribose. Thenucleotide units of the ODN's are interconnected by a “phosphate”backbone, as is well known in the art. The ODNs of the ODN-MGBconjugates of the present invention may include, in addition to the“natural” phosphodiester linkages, phosphorothiotes andmethylphosphonates.

The ODNs of the ODN-MGB conjugates of the present invention may alsohave a relatively low molecular weight “tail moiety” attached to eitherat the 3′ or 5′-end. The “tail moiety” in this particular context is tobe distinguished from the minor groove binding moiety, which ispreferably also attached to the 3′ or 5′ ends, or to both. Thus, in thiscontext the “tail moiety” if present at all, is attached to the end ofthe ODN which does not bear the minor groove binder moiety. By way ofexample, a tail molecule may be a phosphate, a phosphate ester, an alkylgroup, and aminoalkyl group, or a lipophilic group.

With regard to the possible variations of the nucleotide units, the“phosphate backbone” and “tail” of the ODNs of the ODN-MGB conjugates ofthe present invention, the following should be kept in mind. Theprincipal useful action of the ODN-MGB conjugates of the presentinvention lies in the ability of the ODU portion of the molecule to bindto a complementary sequence in single stranded DNA, RNA, double strandedDNA, and DNA—RNA hybrid, in a manner in which the minor groove bindingmoiety is incorporated in the newly formed “duplex” and therebystrengthens the bond, that is, increases the melting temperature (andassociation constant) of the newly formed duplex. Additionally, thosepreferred embodiments of the ODN-MGB conjugates of the present inventionwhich include a cross-linking agent, also result in permanent covalentattachment of the ODN-MGB molecule to the complementary DNA or RNAstrand, resulting in a permanently bound form. In light of theforegoing, those skilled in the art will readily understand that theprimary structural limitation of the various component parts of the ODNportion of the ODB-MGB conjugate of the present invention lies only inthe ability of the ODN portion to form a complementary strand to anyspecific target sequence, and that a large number of structuralmodifications, per se known in the art, are possible within thesebounds. Moreover, synthetic methods for preparing the variousheterocyclic bases, nucleosides, nucleotides and oligonucleotides whichcan form the ODN portion of the ODN-MGB conjugates of the presentinvention, are generally speaking well developed and known in the art.N₄,N₄-ethano-5-methyldeoxycytidine, its nucleoside, nucleotide and/oroligonucleotides incorporating this base can be made in accordance withthe teachings of Webb, T. R.; Matteucci, M. D. Nucleic Acids Res., 1986,14, 7661-7674, Webb, T. R.; Matteucci, M. D. J. Am. Chem. Soc., 1986,108, 2764. 4-aminopyrazolo[3,4-d]pyrimidine,6-amino-4-hydroxypyrazolo[3,4-dpyrimidine, their nucleosides,nucleotides and oligonucleotides incorporating this base can be made inaccordance with the teachings of Kazimierczuk et al. J. Am. Chem. Soc.,1984, 106, 6379-6382. Whereas oligonucleotide synthesis, in order toprepare an ODN of specific predetermined sequence so as to becomplementary to a target sequence, can be conducted in accordance withthe state of the art, a preferred method is described below. Thepreferred method incorporates the teaching of U.S. application Ser. No.08/090,408 filed on Jul. 12, 1993, which has been allowed and the issuefee was paid. The specification of U.S. application Ser. No. 08/090,408is hereby expressly incorporated by reference.

The linking group is a moiety which covalently links the ODN portion ofthe conjugate to the minor groove binder moiety. Preferably, the linkinggroup is such that the linkage occurs through a chain of no more than 15atoms. Also preferably in accordance with the present invention theminor groove binder moiety is covalently attached to either the 3′ or 5′end of the oligonucleotide. Nevertheless, attachment to a nucleotide inintermediate position, and particularly to the heterocyclic base of thenucleotide in intermediate position is also within the scope of theinvention. Generally speaking, the linking group is derived from abifunctional molecule so that one functionality such as an aminefunctionality is attached for example to the phosphate on the 5′ end ofthe ODN, and the other functionality such as a carbonyl group (CO) isattached to an amino group of the minor groove binder moiety.Alternatively, the linking group may be derived from an amino alcohol sothat the alcohol function is linked, for example, to the 3′-phosphateend of the ODN and the amino function is linked to a carbonyl group ofthe minor groove binder moiety. Still another alternative of a linkinggroup includes an aminoalcohol (attached to the 3′-phosphate with anester linkage) linked to an aminocarboxylic acid which in turn is linkedin a peptide bond to the carbonyl group of the minor groove binder.Thus, preferred embodiments of the linking group have the formulas—HN(CH₂)_(m)CO, O(CH₂)_(m)CO and (CH₂)_(m)CH(OH)(CH₂)_(m)NHCO(CH₂)_(m)NH where the limitation on m is that the minorgroove binder moiety should not be separated by more than approximately15 atoms from the ODN. Preferred embodiments of linking groups are—O(CH₂)₆NH, —OCH₂CH(OH)CH₂NHCOCH₂CH₂NH and —HN(CH₂)₅CO. As it was notedabove, the linking group could also be conceptualized as part of theminor groove binder moiety, which in that case would be considereddirectly attached to the ODN.

The basic limitation for the minor groove binder moiety has been setforth above, and is not definable by specific chemical structure. Inaddition to the molecular structure which causes minor groove binding,the minor groove binder moiety may also carry additional functions, aslong as those functions do not interfere with minor groove bindingability. For example a reporter group, which makes the minor groovebinder readily detectable by color, uv. spectrum or other readilydiscernible physical or chemical characteristic, may be covalentlyattached to the minor groove binder moiety. An example for such areporter group is a diazobenzene function which in the example of apreferred embodiment is attached to a carbonyl function of the minorgroove binder through a —HN(CH₂)_(m)COO(CH₂)_(m)S(CH₂)_(m)—bridge.Again, the reporter group or other like function carried by the minorgroove binder can also be conceptualized as part of the minor groovebinder moiety itself.

Preferred embodiments of the ODN-MGB conjugates are defined by thefollowing chemical Formula 1. This definition includes the preferredembodiments of the minor groove binder moiety in accordance with thepresent invention, which may also include all or part of the linkinggroup and other appendant groups such as a reporter group, as discusedabove:

where

x is O or S;

q is an integer between 3 to 100;

R₈ is H, OH, alkoxy having 1 to 6 carbons, O—C₂-C₆alkenyl, or F;

B is an aglycon selected from a group consisting of a heterocyclic basenaturally found in nucleic acids and hypoxanthine, 2-aminoadenine,2-thiouracil, 2-thiothymine, 5-N ⁴-ethenocytosine, 4-aminopyrrazolo[3,4-d]pyrimidine, 6-amino-4-hydroxy-[3,4-d]pyrimidine;

W₁ is H, PO(OH)₂ or a salt thereof, or a minor groove binder moietyattached to the 3′ or 5′ end of said oligonucleotide, the W₁ groupincluding the linking group which covalently binds the minor groovebinder moiety to the oligonucleotide through no more than 15 atoms,;

W₂ is absent or is a minor groove binder moiety attached to one of theaglycons B, the W₂ group including the linking group which covalentlybinds the minor groove binder moiety to said aglycon, or W₂ is across-linking functionality including a linker arm which covalentlybinds the cross-linking functionality to said aglycon,

wherein the minor groove binder moiety is a radical of a molecule havinga molecular weight of approximately 150 to approximately 2000 Daltonsthat bind in a non-intercalataing manner into the minor groove of doublestranded DNA, RNA or hybrids thereof with an association constantgreater than approximately 10³, with the proviso that at least one ofsaid W₁ and W₂ groups is a minor groove binder moiety; and

wherein further the minor groove binder moiety including the linkinggroup has the formula selected from the group consisting of groups (a),(b), (c), (d) and (e):

R₁—(HN—Y₁—CO)_(n)-R₂  (a)

where Y₁ represents a 5-membered ring having two double bonds and 0 to 3heteroatoms selected from the group consisting of N, S and O, the NH andCO groups are attached respectively to two ring carbons which areseparated by one ring atom from one another, the ring atom positionedbetween said two ring carbons is substituted only with H or isunsubstituted, each of the remaining ring atoms may be optionallysubstituted with 1, 2 or 3 R₃ groups;

R₁—(R₆N—Y₂—CO)_(n)—R₂  (b)

where Y2 is a ring system consisting of a 6-membered aromatic ringcondensed with a 5-membered ring having one double bond, the condensedring system having 0 to 3 heteroatoms selected from the group consistingof N, S and O, each of the R₆N and CO groups is attached to a ringcarbon which is in a different ring of the condensed ring system, andwhich is the second ring atom, respectively, from one common bridgeheadring atom, the CO and NR₆ groups thereby positioning 2 non-bridgeheadring atoms between themselves on one side and 3 non-bridgehead ringatoms on the other side of the condensed ring system, the twonon-bridgehead ring atoms on the one side being optionally substitutedwith an R₇ group, the three non-bridgehead ring atoms on the other sideof the condensed ring system being optionally substituted with an R₃group;

R₁—(CO-Y₃—NH) )_(n)—R₂  (c)

where Y₃ is a 6-membered aromatic ring having 0 to 3 N heteroatoms, andwhere each of the CO and NH groups is attached to a ring carbon, saidring carbons being in 1,4 position relative to one another, two ringatoms not occupied by the CO or NH groups on either one of the two sidesof the 6-membered ring being optionally substituted with an R₃ group,the two ring atoms not occupied on the other side of the 6 membered ringbeing optionally substituted with an R₇ group;

R₁—(HN—Y₄—HN—CO—Y₄—CO)_(p)—R₂  (d)

where Y₄ is a 6-membered aromatic ring having 0 to 3 N heteroatoms, andwhere each of the CO and NH groups is attached to a ring carbon, saidring carbons being in 1,4 position relative to one another in each ring,two ring atoms not occupied by the CO or NH groups on either one of thetwo sides of the 6-membered ring being optionally substituted with an R₃group, the two ring atoms not occupied on the other side of the 6membered ring being optionally substituted with an R₇ group;

R₁—(Y₅)_(n)—R₂  (e)

where Y₅ is a ring system consisting of a 6-membered aromatic ringcondensed with a 5-membered ring having one double bond, the condensedring system having 0 to 3 heteroatoms selected from the group consistingof N, S and O, each of the R₁ and R₂ groups is attached to a ring carbonwhich is in a different ring of the condensed ring system, and which isthe second ring atom, respectively, from one common bridgehead ringatom, the R₁ and R₂ groups thereby positioning 2 non-bridgehead ringatoms between themselves on one side and 3 non-bridgehead ring atoms onthe other side of the condensed ring system, the two non-bridgehead ringatoms on the one side being optionally substituted with an R₇ group, thethree non-bridgehead ring atoms on the other side of the condensed ringsystem being optionally substituted with an R₃ group;

where R₁ and R₂ independently are H, F, Cl, Br, I, NH₂, NHR₄, N(R₄)₂,N(R₄)₃ ⁺, OH, —O—, —S—, OR₄, SH, SR₄, COR₄, CONHR₄, CON(R₄)₂, R₄,H₂N(CH₂)_(m)CO, CONH₂, CONHR₄,H₂N(CH₂)_(m)COO(CH₂)_(m)S(CH₂)_(m)C₆H₄NNC₆H₄, —HN(CH₂)_(m)CO, —CONH—,—CONR₄, —HN(CH₂)_(m)COO(CH₂)_(m)S(CH₂)_(m)C₆H₄NNC₆H₄, and—(CH₂)_(m)CH(OH) (CH₂)_(m)NHCO(CH₂)_(m)NH—, or one of the R₁ and R₂groups is absent;

R₃ is selected from the group consisting of F, Cl, Br, I, NH₂, NHR₄,N(R₄)₂, N(R₄)₃ ⁺, OH, OR₄, SH, SR₄, COR₄, CONHR₄, CON(R₄)₂ and R₄, orthe R₃ groups may form a 3, 4, 5 or 6 membered ring condensed to the Y₁ring;

R₄ is an alkyl or cycloalkyl group having 1 to 20 carbons, an alkenyl orcycloalkenyl group having 1 to 20 carbons and 1 to 3 double bonds, acarbocyclic aromatic group of no more than 25 carbons, a heterocyclicaromatic group of no more than 25 carbons, a carbocyclic or heterocyclicarylalkyl group of no more than 25 carbons, where R₄ may be optionallysubstituted with 1, 2 or 3 F, Cl, Br, I, NH₂, NHR₅, N(R₅)₂, N(R₅)₃ ⁺,OH, OR₅, SH, SR₅, COR₅, CONHR₅, CON(R₅)₂ or R₅ groups;

R₅ is alkyl of 1 to 6 tarbons,

R₆ is H, alkyl of 1 to 5 carbons, or R₆ and R₇ jointly form a 4, 5, or 6membered ring, optionally an —O—, —S—, —NH—, —NCH₃—, or N-lower alkylgroup being part of said ring;

R₇ is F, methyl or ethyl; —CH₂—, or —CH₂CH₂—;

m is an integer between 1 to 10;

n is an integer between 1 to 10, and

p is an integer between 1 to 5.

Still more preferred embodiments of the ODN-MBG conjugates of thepresent invention are those where the minor groove binder moiety isdefined as follows:

(1) the minor groove binding moiety is represented by formula (a) aboveand the five membered ring has the structure

(2) the minor groove binding moiety is represented by formula (a) abovewherein the five membered ring has the structure

 and

(3) the minor groove binding moiety is represented by formula (b) andthe condensed ring system has the structure

Embodiments Containing a Cross-linking Functionality

A class of preferred embodiments of the ODN-MGB conjugates of thepresent invention also include one or more cross-linking functionalitieswhereby after the ODN-MGB conjugate is bound to a complementary targetsequence of DNA, RNA or fragment thereof, the cross-linkingfunctionality irreversibly reacts with the target and forms a covalentbond therewith. Advantages of such covalent linking to a target sequenceare in analytical, diagnostic use, as in hybridization probes, and intherapeutic (anti-sense and anti-gene) applications. The minor groovebinder moiety which is also covalently bound to the ODN that complementsthe target sequence, enhances the initial non-covalent binding of theODN-MGB conjugate to the target sequence and therefore facilitates thesubsequent covalent bonding through the cross-linking function. Thefollowing considerations are pertinent as far as the cross-linkingfunctionalities or agents incorporated into this class of ODN-MGBconjugates are concerned.

The cross-linking agents incorporated in the present invention arecovalently bonded to a site on the ODN-MGB. Its length and stericorientation should be such that it can reach a suitable reaction site inthe target DNA or RNA sequence after the ODN-MGB is hybridized with thetarget. By definition, the cross-linking functionality or agent has areactive group which will react with a reactive group of the target DNAor RNA sequence. The cross-linking agent (or agents) may be covalentlyattached to one or more of the heterocyclic bases, to the sugar ormodified sugar residues, or to the phosphate or modified phosphatefunctions of the ODN-MGB conjugates. The cross-linking agent may also beattached to the minor groove binder moiety as long as it does notinterfere with its minor groove binding ability. Preferably thecross-linking agent or functionality is attached to one of theheterocyclic bases.

In simple terms the cross-linking agent itself may conceptually bedivided into two groups or moieties, namely the reactive group, which istypically and preferably an electrophilic leaving group (L), and an“arm” (A) which attaches the leaving group L to the respective site onthe ODN-MGB. The leaving group L may be chosen from, for example, suchgroups as chloro, bromo, iodo, SO₂R′″, or S⁺R′″R″″, where each of R′″and R″″ is independently C₁₋₆ alkyl or aryl or R′″ and R″″ together forma C₁₋₆ alkylene bridge. Chloro, bromo and iodo are preferred. Withinthese groups haloacetyl groups such as —COCH₂I, and bifunctional“nitrogen mustards”, such as —N—[(CH₂)₂—Cl]₂ are preferred. The leavinggroup will be altered by its leaving ability. Depending on the natureand reactivity of the particular leaving group, the group to be used ischosen in each case to give the desired specificity of the irreversiblybinding probes.

Although as noted above the “arm” (or linker arm) A may conceptually beregarded as a single entity which covalently bonds the ODN-MGB to theleaving group L, and maintains the leaving group L at a desired distanceand steric position relative to the ODN-MGB, in practice the “arm” A maybe constructed in a synthetic scheme where a bifunctional molecule iscovalently linked to the ODN-MGB, or to the ODN before the minor groovebinder moiety is attached (for example by a phosphate ester bond to the3′ or 5′ terminus, by a carbon-to-carbon bond to a heterocyclic base orby carbon to nitrogen bond to an amino substituted heterocyclic base)through its first functionality, and is also covalently linked throughits second functionality (for example an amine) to a “hydrocarbylbridge” (alkyl bridge, alkylaryl bridge or aryl bridge, or the like)which, in turn, carries the leaving group L.

A general formula of the cross linking function is thus —A—L, or —A—L₂where L is the above defined leaving group and A is a moiety that iscovalently linked to the ODN-MGB. The A “arm” moiety itself should beunreactive (other than through the leaving group L) under the conditionsof hybridization of the ODN-MGB with the target sequence, and shouldmaintain the leaving group L in a desired steric position and distancefrom the desired site of reactions such as an N-7 position of aguanosine residue in the target sequence. Generally speaking, the lengthof the A group should be equivalent to the length of a normal alkylchain of approximately 2 to 20 carbons.

An examplary more specific formula for a class of preferred embodimentsof the cross-linking function is

—(CH₂)_(q)—Y—(CH₂)_(m)—L,

where L is the leaving group, defined above, each of m and q isindependently 0 to 8, inclusive, and where Y is defined as a “functionallinking group”. For clarity of description this “functional linkinggroup” is to be distinguished from the “linking group” that attaches theminor groove binder moiety to the ODN, although the functional linkinggroups described here for attaching the cross-linking agent can also beused for attaching a minor groove binder moiety to either end of theODN, or to a nucleotide in intermediate position of the ODN. A“functional linking group” is a group that has two functionalities, forexample —NH₂ and —OH, or —COOH and —OH, or —COOH and —NH_(2,) which arecapable of linking the (CH₂)_(q) and (CH₂)_(m) bridges. An acetylenicterminus (HC≡C—) is also a suitable functionality for Y, because it canbe coupled to certain heterocycles, as described below.

Other examplary and more specific formulas for a class of preferredembodiments of the cross-linking function are

—(CH₂)_(q)—NH—CO—(CH₂)_(m)—(X)_(n)—N(R₁)—(CH₂)_(p)—L

and

—(CH₂)_(q′)—O—(CH₂)_(q″)—H—CO—(CH₂)_(m)—(X)_(n)—N(R₁)—(CH₂)_(p)—L

where q, m and L are defined as above in connection with the descriptionof the cross-linking functions, q′ is 3 to 7 inclusive, q″ is 1 to 7inclusive, X is phenyl or simple substituted phenyl (such as chloro,bromo, lower alkyl or lower alkoxy substituted phenyl), n is 0 or 1, pis an integer from 1 to 6, and R₁ is H, lower alkyl or (CH₂)_(p), —L.Preferably p is 2. Those skilled in the art will recognize that thestructure —N(R₁)—(CH₂)₂—L describes a “nitrogen mustard”, which is aclass of potent alkylating agents. Particularly preferred are withinthis class of ODN-MGB conjugates those where the cross-linking agentincludes the functionality—N(R₁)—(CH₂)₂—L where L is halogen, preferablychlorine; and even more preferred are those ODN-MGB conjugates where thecross linking agent includes the grouping—N—((CH₂)₂—L]₂ ( a“bifunctional” N-mustard).

A particularly preferred partial structure of the cross linking agentincludes the grouping

—CO—(CH₂)₃ —C₆H₄—N—[(CH₂)₂Cl]₂.

In a preferred embodiment the just-noted cross-linking group is attachedto an n-hexylamine bearing tail at the 5′ and 3′ ends of the ODN inaccordance with the following structure:

R′—O—(CH₂)₆ —NH—CO—(CH₂)₃—C₆H₄—N—[(CH₂)₂Cl]₂

where R′ signifies the terminal 5′ or 3′-phosphate group of the ODN. Theother terminal, or a nucleotide in an intermediate position bears theminor groove binder moiety.

In accordance with other preferred embodiments, the cross-linkingfunctionality is covalently linked to the heterocyclic base, for exampleto the uracil moiety of a 2′-deoxyuridylic acid building block of theODN-MGB conjugate. The linkage can occur through the intermediacy of anamino group, that is, the “arm-leaving group combination” (A—L) may beattached to a 5-amino-2′-deoxyuridylic acid building unit of the ODN. Instill other preferred embodiments the “arm-leaving group combination”(A—L) is attached to the 5-position of the 2′-deoxyuridylic acidbuilding unit of the ODN by a carbon-to-carbon bond. Generally speaking,5-substituted-2′-deoxyuridines can be obtained by an adaptation of thegeneral procedure of Robins et al. (Can. J. Chem., 60:554 (1982); J.Org. Chem., 48:1854 (1983)). In accordance with this adaptation,palladium-mediated coupling of a substituted 1-alkyne to5-iodo-2′-deoxyuridine gives an acetylene-coupled product. Theacetylenic durd analog is reduced, with Raney nickel for example, togive the saturated compound, which is then used for direct conversion toa reagent for use on an automated DNA synthesizer. Examples of reagentswhich can be coupled to 5-iodo-2′-deoxyuridine in accordance with thismethod are HC≡CCH₂OCH₂CH₂N(CO)₂C₆H₄ (phtalimidoethoxypropyne) andHC≡CCH₂OCH₂CH₂NHCOCF₃ (trifluoroacetamidoethoxypropyne).

In these examples the nucleosides which are obtained in this scheme areincorporated into the desired ODN, and the alkylating portion of thecross-linking agent is attached to the terminal amino group only afterremoval of the respective phtalic or trifluoroacetyl blocking groups.Other examples of nucleotides where the crosslinking agent is attachedto a heterocyclic base, are 2′-deoxy-4-aminopyrazolo[3,4-d]pyrimidinederivatives. These compounds can be made in accordance with the teachingof published PCT application WO: 90/03370 (published on Apr. 5, 1990).

Discussing still in general terms the structures of the modified ODNs ofthe present invention, it is noted that examination of double-strandedDNA by ball-and-stick models and high resolution computer graphicsindicates that the 7-position of the purines and the 5-position of thepyrimidines lie in the major groove of the B-form duplex ofdouble-stranded nucleic acids. These positions can be substituted withside chains of considerable bulk without interfering with thehybridization properties of the bases. These side arms may be introducedeither by derivatization of dThd or dCyd, or by straightforward totalsynthesis of the heterocyclic base, followed by glycosylation. Thesemodified nucleosides may be converted into the appropriate activatednucleotides for incorporation into oligonucleotides with an automatedDNA synthesizer. With the pyrazolo[3,4—d]pyrimidines, which are analogsof adenine, the crosslinking arm is attached at the 3-position, which isequivalent to the 7-position of purine.

The crosslinking side chain (arm=A) should be of sufficient length toreach across the major groove from a purine 7- or 8-position, pyrimidine5-position, pyrrolopyrimidine 5-position or pyrazolopyrimidine3-position and reacting with the N—7 of a purine (preferably guanine)located above (on the oligomer 3′-side) the base pair containing themodified analog. The crosslinking side chain (arm=A) holds thefunctional group away from the base when the base is paired with anotherwithin the double-stranded complex. As noted above, broadly the arm Ashould be equivalent in length to a normal alkyl chain of 2 to 20carbons. Preferably, the arms include alkylene groups of 1 to 12 carbonatoms, alkenylene groups of 2 to 12 carbon atoms and 1 or 2 olefinicbonds, alkynylene groups of 2 to 12 carbon atoms and 1 or 2 acetylenicbonds, or such groups substituted at a terminal point with nucleophilicgroups such as oxy, thio, amino or chemically blocked derivativesthereof (e.g., trifluoroacetamido, phthalimido, CONR′, NR′CO, andSO₂NR′, where R′=H or C₁₋₆alkyl). Such functionalities, includingaliphatic or aromatic amines, exhibit nucleophilic properties and arecapable of serving as a point of attachment to such groups as

—(CH₂)_(m)—L,

and

—CO—(CH₂)_(m)—(X)_(n)—N(R₁)—(CH₂)_(p)—L

which are described above as components of examplary cross-linkingfunctional groups.

After the nucleoside or nucleotide unit which carries the crosslinkingfunctionality A—L, or a suitable precursor thereof, (such as the—(CH₂)_(q)—NH₂ or —(CH₂)_(q)—Y group, where Y terminates with anucleophilic group such as NH₂) is prepared, further preparation of themodified oligonucleotides of the present invention can proceed inaccordance with state-of-the-art. Thus, to prepare oligonucleotides,protective groups are introduced onto the nucleosides or nucleotides andthe compounds are activated for use in the synthesis ofoligonucleotides. The conversion to protected, activated forms mayfollow the procedures as described for 2′-deoxynucleosides in detail inseveral reviews. See, Sonveaux, Bioorganic Chemistry, 14:274-325 (1986);Jones, in “Oligonucleotide Synthesis, a Practical Approach”, M. J. Gait,Ed., IRL Press, p. 23-34 (1984).

The activated nucleotides are incorporated into oligonucleotides in amanner analogous to that for DNA and RNA nucleotides, in that thecorrect nucleotides will be sequentially linked to form a chain ofnucleotides which is complementary to a sequence of nucleotides intarget DNA or RNA. The nucleotides may be incorporated eitherenzymatically or via chemical synthesis. The nucleotides may beconverted to their5′-O-dimethoxytrityl-3′-(N,N-diisopropyl)phosphoramidite cyanoethylester derivatives, and incorporated into synthetic oligonucleotidesfollowing the procedures in “Oligonucleotide Synthesis: A PracticalApproach”, supra. The N-protecting groups are then removed, along withthe other oligonucleotide blocking groups, by post-synthesis aminolysis,by procedures generally known in the art.

In a preferred embodiment, the activated nucleotides may be useddirectly on an automated DNA synthesizer according to the procedures andinstructions of the particular synthesizer employed. Theoligonucleotides may be prepared on the synthesizer using the standardcommercial phosphoramidite or H-phosphonate chemistries.

A moiety containing the leaving group, such as a haloacyl group, or—CO—(CH₂)_(m)—(X)_(n)—N(R₁)—(CH₂)_(p)—L group (even more preferably aCO—(CH₂)₃—C₆H₄—N—[CH₂CH₂Cl]₂) may be added to the aminoalkyl or liketails (—CH₂)_(q)—Y) following incorporation into oligonucleotides andremoval of any blocking groups.

In the situations where the cross linking agent (A—L moiety) is attachedto the 3′ or 5′ terminus of the oligonucleotide, for example by analkylamine linkage of the formula —(CH₂)_(q)—Y (Y terminating in anamine), the oligonuclotide synthesis may be performed to first yield theoligonucleotide with said aminoalkyl tail, to which then an alkylatingmoiety, such as the above-noted haloacylgroup or—CO—(CH₂)_(m)—(X)n—N(R₁)—(CH₂)_(p)—L is introduced.

An exemplary preferred embodiment of an ODN-MGB conjugate which has across-linking agent attached to one of the nucleotide bases isrepresented by the formula below:

5′-GGTTATTTTTGAAGATACGAATTTCUCCAGAGACACAGCAGGATTTGTCA-CDPI₃ (SEQ IDNO:1) where the underlined symbol “U” (the 26th nucleotide unit in the50mer) represents a 5-(3-aminopropyl)-2′-deoxyuridine which has achlorambucil residue attached to the amino group. The symbol “CDPI₃”represents a minor groove binder moiety as described below in connectionwith Reaction Scheme 1. The 5-(3-aminopropyl)-2′-deoxyuridine componentis incorporated into the ODN by using5′-O-trityl-5-trifluoroacetimidopropyl-2′-deoxyuridine3′-(N,N-diisopropyl-cyanoethyl-phosphoramidite in accordance with theprocedure of Gibson, K. J., & Benkovic, S. J. (1987) Nucleic Acids Res.15, 6455. The chlorambucil residue and the minor groove binder moietyare introduced into the ODN as described in the experimental sectionbelow.

Synthesis of Minor Groove Binder Moieties and ODN-MGB Conjugates

Presently most preferred embodiments of the minor groove binder moietiesof the present invention are “oligopeptides” derived from1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylic acid (CDPI) and from4-amino-N-methylpyrrole-2-carboxylic acid. These are synthetic peptideswhich have repeating units of the structures shown respectively inFormula 2 and Formula 4 where the degree of polymerization (m) of thepeptide is preferably 3 to 5, most preferably 5 for the peptide ofFormula 2 and 3 for the peptide of Formula 4. Reaction Scheme 1discloses a process for preparing a specific tripeptide abbreviated“CDPI₃” which thereafter can be coupled with or without minormodification, to ODNs, to form preferred embodiments of the ODN-MGBconjugates of the present invention.

Referring thus to Reaction Scheme 1, the starting material in thissynthetic scheme is3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylic acid or3-t-butyloxycarbonyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylicacid which can be made in accordance with the chemical literature (D. L.Boger, R. S. Coleman, and B. J. Invergo. J.Org.Chem., 1987, Vol.52,1521-1530). The starting compounds are converted into an active ester bytreatment with the tetrafluorophenyl ester of tri-fluoroacetic acid(TFP-TFA). In compound 1a shown in the scheme the R group is CONH_(2,)in 1b R is t-butyloxycarbonyl (^(t)Boc). The t-butyloxycarbonyl(^(t)Boc) group is a well known protecting group for amino functionswhich can be removed by acid. The resulting activated esters la and lbare reacted with methyl 1,2-dihydro-3H-pyrroloindole-7-carboxylate (alsoavailable in accordance with the chemical literature, see D. L. Boger,R. S. Coleman, and B. J. Invergo. J. Org. Chem., 1987, Vol. 52,1521-1530) to yield the “dimer” peptide compounds 2a and 2c. The methylgroup of the carboxyl function is removed by treatment with base toyield the “dimer” peptides wherein the carboxylic acid group is free.This dimer is activated once more to form an active ester withtetrafluorophenol (2e when R=CONH₂, TFP-CDPI₂; and 2f when R=^(t)Boc,TFP-^(t)Boc-CDPI₂).

After activation with TFP-TFA the active ester of the dimer can be usedfor forming the ODN-MGB conjugate as is described below in connectionwith the corresponding trimer. The activated ester of the dimer peptidecan also be reacted with yet another molecule of methyl1,2-dihydro-3H-pyrroloindole-7-carboxylate to form a “trimer peptide”that has its carboxylic acid function protected as a methyl ester, 3a(methyl 3-carbamoyl-1, 2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylatetrimer). The methyl group is removed by treatment with base and theresulting “trimer peptide” 3b is converted again into an activetetrafluorophenyl ester 3c (2,3,5,6-tetrafluorophenyl3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate trimer,TFP-CDPI₃). The active tetrafluorophenyl ester 3c can be used to furtherlengthen the peptide chain by repeating the steps of reacting withmethyl 1,2-dihydro-3H-pyrroloindole-7-carboxylate, saponifying theresulting methyl ester, and if desired, reacting with TFP-TFA again tomake the active tetrafluorophenyl ester of the peptide incorporating 4CDPI moeieties. As it will be readily understood, these steps can berepeated further until the desired number of CDPI moieties are includedin the peptide. In the herein described preferred embodiments the activetetrafluorophenyl ester of the tripeptide 3c (TFP-CDPI₃) is utilized forcoupling to an ODN to make an ODN-MGB, or for synthesizing an ODN-MGB ona suitable modified controlled pore glass (CPG) solid suport as isdescribed below in connection with Reaction Schemes 4 and 5. ReactionScheme 1 indicates as its last step the preparation of ahydroxylpropylamide derivative from the the active tetrafluorophenylester of the tripeptide 3c (TFP-CDPI₃). The hydroxylpropylamidederivative of the tripeptide 3d(3-carbamoyl-1,2-dihydro-[3H-pyrrolo[3,2-e]indole-7-carbox]-1-amido-3-propanoltrimer, CDPI₃-3-hydroxylpropylamide) can be used for coupling with anODN to obtain an ODN-MGB in accordance with the present invention. Thetripeptide 3d however, was also used as a “free standing” minor groovebinder molecule as a control in certain binding studies which aredescribed below.

Referring now to Reaction Scheme 2 the synthesis of another preferredembodiment of the minor groove binder peptides is disclosed, where the“monomer” is the residue of 4-amino-N-methylpyrrol-2-carboxylic acid,and which embodiment also bears a reporter group/containing adiazobenzene moiety. Thus, in accordance with this scheme6-[(tert-butyloxy)carboxamido]hexanoic acid is condensed in the presenceof N,N-dicyclohexylcarbodiimide with (2-[4-(phenylazo)-benzylthio]-ethanol to form (2-[4-(phenylazo)benzylthio]ethyl5-[(tert-butyloxy) carboxamido]pentylcarboxylate, 11). The ^(t)Bocprotecting group is removed from compound 11 by treatment withtrifluoroacetic acid (TFA) and the resulting compound having a freeamino function is reacted with an activated ester of ^(t)Boc protected4-amino-N-methylpyrrol-2-carboxylic acid. The latter activated estercompound (1,2,3-benzotriazol-1-yl1-methyl-4-(tert-butyloxy)carboxamido-pyrrole-2-carboxylate)is made from1-methyl-4-[tert-butyloxy)carboxamido]pyrrole-2-carboxylic acid which isavailable pursuant to the literature procedure of L. Grehn, V.Ragnarsson, J. Org. Chem., 1981, 46, 3492-3497. The resulting2-[4-(phenylazo)benzylthio]ethyl 5-[1-methyl-4-(tert-butyloxy)carboxamido]pyrrole-2-carboxamido]pentylcarboxylate, 12) hasone unit of the monomer “2-amino-N-methylpyrrol carboxylic acid” residueattached to the reporter group that carries the diazobenzene moiety.After removal of the ^(t)Boc protecting group with trifluoroacetic acidand coupling with one or more molecules of 1,2,3-benzotriazol-1-yl1-methyl-4-(tert-butyloxy)carboxamido-pyrrole-2-carboxylate can beaccomplished, until a peptide containing the desired number of monomerresidues is obtained. Such a compound having n number of monomers and afree amino group is indicated in Reaction Scheme 2 as 16a. Compound 16acan be reacted with an activated ester (such as a1,2,3-benzotriazol-1-yl activated ester) of ^(t)Boc protected6-aminohexanoic acid to provide the oligopeptide shown as compound 16bin Reaction Scheme 2. The ^(t)Boc protecting group can be removed fromthe latter compound under acidic conditions, and the resultingderivative having a free amino function can be attached by conventionalsynthetic methods to either the 3′-phosphate or 5′-phoshate end of anODN. Alternatively, the derivative having a free amino function can alsobe attached to the 3′ or 5′-OH end of an oligonucleotide using a varietyof bifunctional linking groups, as discussed above.

Referring now to Reaction Scheme 3 a general method for coupling a3′-amino tailed or 5′-amino-tailed ODN with the tetrafluorophenyl (TFP)ester activated exemplary minor groove binding oligopeptides isillustrated. Although the scheme shows the use of the TFP activatedexemplary minor groove binding compounds obtained in accordance withReaction Scheme 1, it should be kept in mind that this general method issuitable for the coupling of other TFP activated minor groove bindingcompounds with ODNs, as well. The reference numeral la through 3c inReaction Scheme 3 refer to the exemplary compounds obtained inaccordance with Reaction Scheme 1.

The 3′- or 5′-amino tailed ODNs can be synthesized by conventionalmethods; for example an aminohexyl residue can be attached to either endof the ODN by using commercially available N-monomethoxytritylaminohexylphosphoramidite. Alternatively, the amino tailed ODNs can be synthesizedin accordance with the methods described in U.S. application Ser. No.08/090,408 filed on Jul. 12, 1993 which has been allowed and the issuefee was paid. The specification of U.S. application Ser. No. 08/090,408is expressly incorporated herein by reference. In accordance with thepresent scheme the amino tailed ODN is converted into acetyltrimethylammonium salt to render it soluble in organic solvents,and the tetrafluorophenyl ester activated minor groove binder moleculeis condensed therewith, preferably in DMSO as a solvent.

Reaction Scheme 4 discloses another method of coupling an active esterof a minor groove binder molecule to a 5′-amino tailed ODN. The exampleshown in the scheme is that of the TFP ester of the tripeptide derivedfrom 3-carbomoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylic acidresidues (TFP-CDPI₃) but it should be understood that the genericprinciples disclosed in connection with this reaction scheme can be usedwith other minor groove binder molecules as well. In this method, theODN is still attached to a CPG support, and has a free amino group onits “amino tail”. This can be obtained by usingN-monomethoxytritylaminohexyl phosphoramidite mentioned above. Themonomethoxytrityl group is removed after the coupling of thephosphoramidite to give the desired CPG-bearing-“amino-tailed ODN”.Alternatively, such a CPG- can be obtained in accordance with thedisclosure of the above-cited U.S. application Ser. No. 08/090,408, andreferences cited therein. By way of summary, the ODN is synthesizedstepwise attached to the CPG, and having a tail having an amino groupprotected with a 9-fluorenylmethoxycarbonyl (Fmoc) group. After thedesired sequence of nucleotides has been built up, the Fmoc group isremoved from the amino group while the ODN is still attached to the CPGsupport. In accordance with Reaction Scheme 4 of the present inventionthis “CPG-bearing-amino-tailed-ODN” having the free amino group iscondensed with the active ester (TFP-CDPI₃, 3c) or with a like activatedform of a minor groove binder. The ODB-MGB conjugate is thereafterremoved from the CPG support by conventional methods, most frequently bytreatment with ammonia.

Reaction Scheme 5 discloses another preferred method for preparing theODN-MGBs of the present invention. More particularly, Reaction Scheme 5discloses the preferred synthetic process for preparing ODN-MGBs byfirst attaching a linking molecule to a CPG support, thereafterattaching an activated form of minor groove binder to the linkingmolecule, and thereafter building the ODN of desired sequencestep-by-step in an automatic ODN synthesizer using the just describedmodified CPG support. The ODN-MGB conjugate is removed from the CPGsupport only after the ODN moiety of desired sequence has beencompleted. The linking molecule in this case is a trifunctionalmolecule, with each function having different reactivity, which permitattachment to the CPG, reaction with the activated form of minor groovebinder moiety and the building of the ODN portion, each using adifferent functionality of the linking molecule. A more general anddetailed description of this synthetic method and of the trifunctionallinking molecules which can be utilized in the method, but without anyreference to minor groove binders, can be found in U.S. application Ser.No. 08/090,408. Reaction Scheme 5 illustrates this synthetic processwith the example of β-alanilyl-3-amino-1,2-propanediol as thetrifunctional linking molecule, and TFP-CDPI₃ (compound 3c) as theactivated form of the minor groove binder.

Thus in accordance with Reaction Scheme 5, Fmoc protected β-alanine isreacted with tetrafluophenyl trifluoroacetate (TFP-TFA) to provide2,3,5,6-tetrafluorophenyl3-[N-(9-fluorenylmethoxycarbonyl)]aminopropionate (4). The active ester4 is reacted with 3-amino-1,2-propanediol to provide1-[3-[N-(9-fluorenylmethoxycarbonyl)amino]-1-oxopropyl]amino-(R,S)-2,3-propanediol(5). The primary hydroxyl group of 5 is thereafter protected with adimethoxytrityl group to give1-[3-[N-(9-fluorenylmethoxycarbonyl)-amino]-1-oxopropyl]amino-(R,S)-2-[[bis(methoxyphenyl)phenylmethoxy]metyl]-2-ethanol(6). The secondary hydroxyl group of compound 6 is reacted with succinicanhydride and the carboxylic group in the resulting compound isthereafter converted into an active ester, 2,3,5,6-tetrafluorophenyl1-[3-[N-(9-fluorenylmethoxycarbonyl)amino]-1-oxopropyl]amino-(R,S)-2-[[bis(methoxyphenyl) phenylmethoxy]metyl]-2-ethyl butanedioate (7). Compound 7 is then attached to a longchain aminoalkyl controlled pore glass support (LCAA-CPG, or alkylamineCPG) which is commerciallly available and is described in theabove-cited U.S. application Ser. No. 08/090,408. The resulting“modified CPG” is shown in Reaction Scheme 5 as Compound 8. The Fmocprotecting group is removed from 8 by treatment with mild base(piperidine in dimethylformamide) to yield the “modified CPG” 9 that hasa free primary amine function as part of the linking molecule. In thenext step the activated minor groove binder molecule, in this instanceTFP-CDPI₃ (compound 3c) is reacted with the primary amine function of 9,to yield the modified CPG 10 that includes the minor groove bindermoiety and still has the primary hydroxyl group of the linking groupprotected with a dimethoxytrityl group. Although this is not shown inReaction Scheme 5, in the subsequent steps the dimethoxytrityl group isremoved and the ODN synthesis is performed in an automatic synthesizer,by steps which are now considered conventional in the art. When thesynthesis is complete the ODN-YGB conjugate is removed from the CPGsupport by treatment with ammonia. The latter step cleaves the bondattaching the secondary hydroxyl group of the 3-amino-1,2-propanediolmoiety to the CPG support.

Biological Testing and Discussion

The ODN-MGB conjugates bind to single stranded DNA. They also bind todouble stranded DNA in the presence of a recombinase enzyme, and in somecases to single stranded RNA and DNA and RNA hybrids as well. Thebinding however occurs only if the ODN,moiety is complementary, orsubstantially complementary in the Watson-Crick sense, to a targetsequence in the target DNA or RNA. When this condition is met, thebinding of the ODN-MGB to the target sequence is significantly strongerthan binding of the same ODN would be without the minor groove binder.The foregoing is demonstrated by the tests described below, and providesutility for the ODN-MGB conjugates of the present invention asanalytical and diagnostic hybridization probes for target DNA or RNAsequences, and in therapeutic antisense and anti-gene applications.

TABLE 1 T_(m)'s data of the (dAP)₈ + (dTP)₈ duplex carryingintercalators or oligo-(1-methyl-2-carboxy-4amino) pyrrole residuesattached to 3′-end of the ODN.^(a) COMPLEX T_(m) ΔT_(m) ^(b) (dAp)₈ +(dTp)₈ 21.1 — (dAp)₈ + (dTp)₈ + Distamycin A^(c) 47.1 26.0 (dAp)₈ +(dTp)₈ − X_(m) m = 2 39.4 18.3 m = 3 51.7 30.6 m = 4 60.2 39.1 m = 565.4 44.3 (dTp)₈ + (dAp)₈ −X_(m) m = 2 29.1 8.0 m = 3 39.0 17.9 m = 442.7 21.6 m = 5 52.6 31.5 (dAp)₈ − Y + (dTp)₈ 30.5 9.4 (dAp)₈ − Y +(dTp)₈ − Y^(d) 42.9 21.8 ^(a)Reported parameters are averages of atleast three experiments. Optical melts were conducted in 0.2 M NaCl, 0.1mM EDTA, 0.01 M (±0.1° C.) Na₂HPO₄, pH 7.0 with [(dTp)₈ · (dAp)₈] = 2.5· 10⁻⁵ M. ^(b)The difference in T_(m) between modified and unmodifiedduplexes. ^(c)Concentration of distamycin A was 2.5 · 10⁻⁵ M.^(d)Ethidium bromide (EtBr) was conjugated by its 8-NH₂-position to the3′-terminal phosphate of the ODNs through a β-alanine linker by themethod in ref 12.

Table 1 illustrates the melting temperature of several complexes formedof complementary oligonucleotides which have the minor groove bindermoiety derived from 4-amino-N-methylpyrrol-2-carboxylic acid residues.The minor groove binder moiety is specifically shown as the radical X bythe formula below Table 1. It is noted that the radical X also includesa linking moiety which is derived from 6-aminohexanoic acid. Theoligonucleotides utilized here are 8-mers of 2′-deoxyadenylic acid, and8-mers of thymidylic acid. The minor groove binder X is attached to theODNs at the 3′-phosphate end, the 5′-end of these ODNs have nophosphate. In this regard it is noted that the ODNs are abbreviated inthese and the other tables in the manner customary in the art. The groupY symbolizes an ethidium bromide moiety attached to the 3′phosphate endthrough a “β-alanine” linking moiety. The Y group represents anintercalating group and acts as a control for comparison with the minorgroove binding groups. The symbol m represents the number of4-amino-N-methylpyrrol-2-carboxylic acid residues present in eachODN-MGB of the table.

As is known in the art, the melting temperature (T_(m)) of anoligonucleotide or polynucleotide duplex is defined as that temperatureat which 50% of the respective oligonucleotide or polynucleotide isdissociated from its duplex, Watson Crick hydrogen bonded form. A highermelting temperature (T_(m)) means a more stable duplex. As is knownfurther, the melting temperature of an oligonucleotide or polynucleotideis dependent on the concentration of the nucleotide in the solution inwhich the melting temperature is measured, with higher concentrationsresulting in higher measured melting temperatures. The meltingtemperatures indicated in these tables were measured under conditionsindicated in the table and in the experimental section. ▴T_(m)represents the change in melting temperature of the modified duplexrelative to the melting temperature of the (dAp)₈.(dTp)₈ complex whichhas no minor groove binder moiety.

As it can be seen from Table 1, the covalently bound minor groove bindermoiety significantly increases the stability (melting temperature Tm) ofthe complex, whether the group X (minor groove binder moiety) isattached to the (dTp)₈ or to the (dAp)₈ oligonucleotide. In thisinstance the greatest degree of stabilization (highest meltingtemperature) is achieved when the minor groove binder moiety is a 5-meroligopeptide. In the comparative experiment when the intercalating groupY is attached to the (dAp)₈ oligomer, a comparatively much smallerdegree of stabilization is attained. Even attaching the intercalating Ygroup to each of the two strands of oligomers in this experiment, raisedthe melting temperature less than the minor groove binder moiety havingfive 4-amino-N-methylpyrrol-2-carboxylic acid residues.

TABLE 2 T_(m)'s data of the duplexes formed by hexadeca-,octathymidylate and their oligo-(1- methyl-2-carboxy-4amino) pyrrolederivatives with polydeoxyriboadenylic acid in 0.2 M NaCl, 0.01 MNa₂HPO₄, 0.1 mM EDTA (pH 7.0). X is same as Table 1. Oligo DerivativeT_(m) ° C. ΔT_(m) ° C. SEQ ID NO: (dTp)₁₆ 48.5 — 2 (dTp)₁₆-NH(CH₂)₆COOH49 0.5 3 (dTp)₁₆-X m = 1 49.3 0.8 4 m = 2 55.6 7.1 5 m = 3 61 12.5 6 m =4 66 17.5 7 m = 5 68 19.5 8 (dTp)₈ 28 — — (dTp)₈-X m = 1 28 0 — m = 2 4012 — m = 3 52 24 — m = 4 60 32 — m = 5 66 38 —

Table 2 discloses information in a manner similar to Table 1. In thetests reported in this table 16-mer ODNs of thymidylic acid having theminor groove binder moiety represented by X (X is the same as inTable 1) were complexed with polydeoxyriboadenylic acid. As acomparative control a 16 mer ODN of thymidylic acid (dTp)₁₆ (SEQ IDNO:2) connected at its 3′-phosphate end to 6-aminohexanoic acid was alsotested. Additionally an 8-mer of thymidylic acid (dTp)₈ and itsconjugates with the minor groove binders of varying peptide length werealso tested. In these tests too, the minor groove binder attached to theODN causes significant stabilization of the complex between the ODN-MGBand the complementary DNA strand. Greatest stabilization occurs when thenumber of 4-amino-N-methylpyrrol-2-carboxylic acid residues in the minorgroove binder moiety is five. In contrast, the aminohexanoic acid tailon the 16-mer ODN results in virtually no stabilization of the complex.

TABLE 3 Melting temperatures (° C.) of duplexes formed by poly (dA) andpoly (rA) with (Tp)₈ strands terminally linked to CDPI₁₋₃ and BocDPI₁₋₂ligands.^(a) Octathymidylate poly (dA) poly (rA) derivative T_(m) ΔT_(m)T_(m) ΔT_(m) (dTp)₇dTp-L1 25 — 13 — (dTp)₇dTp-L1-X m = 1 34  9 18 5(dTp)₇dTp-L1-X m = 2 50 25 —^(b) — (dTp)₇dTp-L1-X m = 3 68 (65) 43 (40)32 (31) 19 (18) (dTp)₇dTp-L1-Y m = 1 26  1 12 −1  (dTp)₇dTp-L1-Y m = 243 18 17 4 L1-pdT(pdT)₇ 24 — 12 — X-L1-pdT(pdT)₇ m = 1 31  7 14 2X-L1-pdT(pdT)₇ m = 2 49 25 —^(b) — X-L1-pdT(pdT)₇ m = 3 68 44 35 23 Y-LI-pdT(pdT)₇ m = 1 23 −1  9 −3  Y-L1-pdT(pdT)₇ m = 2 41 17 19 7^(a)The data in brackets were obtained for the derivative with linkerL2. ^(b)No melting transition was observed.

L1 = —O(CH₂)₆NH— L2 = —OCH₂CH(OH)CH₂NHCOCH₂CH₂NH—

Table 3 discloses melting temperature (T_(m)) and change in meltingtemperature (▴T_(m)) data in tests where the oligonuclotide is an 8-merof thymidylic acid having a minor groove binder moiety attached to iteither at the 5′-phosphate or 3′-phosphate end, as indicated in thetable. The minor groove binder moieties represented here by X and Y are“oligopeptides” based on the residue of1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylic acid (CDPI or BocDPI)and their structures are shown in the table. These minor groove bindingoligopeptides are attached to the ODN through a linking moiety “L1 orL2” the structures of which are also shown below the table. The ODN-MGBconjugates were incubated with a complementary ribo- or deoxyribohomopolymer. Thus for ODN-MGB conjugates comprising ODNs of thymidylicacid, poly A or poly-da was used. The change in melting temperature(▴T_(m)) is indicated relative to the complex with the ODN which bearsthe corresponding linking group L1 or L2 in the corresponding end of theODN, but bears no minor groove binding moiety. As it can be seen fromTable 3, these ODN-MGB complexes again exhibit significant stabilizationof the complex with the complementary deoxyribo homopolymer, with thegreatest stabilization occurring in these tests when the minor groovebinding moiety has 3 CDPI units. Surprisingly, stabilization of thecomplex occurs even when the ODN-MGB is incubated with a complementaryribohomopolymer. This is surprising because it had been generallyobserved in the prior art that free standing minor groove bindingmolecules do not bind to DNA-RNA hybrids.

TABLE 4 T_(m)'s data (° C.) of heterogeneous duplexes phosphodiester andphosphorothioale backbones and oligo (pyrroloindole carboxamide) peptideresidues attached to the different positions^(a). Derivative ofCpApTpCpCpGpCpT Derivative of ApGpCpGpGpApTpG Type of DNA terminal3′-L2-X & 2′-DNA PS^(a) Type of Backbone modification 3′-L1- 3′-L2-X5′-X-L1 5′-X-L1 none^(b) 5′-X-L1- 3′-L2-X DNA 3′-L1- 41 52 45 50 33 2740 3′-L2-X 57 81 78 77 50 73 77 5′-X-L1- 58 79 76 76 49 70 75 3′-L2-X5′-X-L1- 60 72 — 65 — — — 2′-DNA PS^(c) none^(b) 32 43 32 — 24 16 285′-X-L1 38 69 67 — 28 62 63 3′-L2-X 45 74 71 — 36 64 69^(a)Concentration of ODNs in the melting mixtures was 2 × 10⁻⁴ M in 140mM KCl, 10 mM MgCl₂, 20 mM HEPES-HCl (pH 7.2). ^(b)The ODN has free 3′-and 5′-OH groups. ^(c)PS is phosphorothioate linkage

L1 = —O(CH₂)₆NH— L2 = —OCH₂CH(OH)CH₂NHCOCH₂CH₂NH—

Table 4 discloses results of a study of duplex formation betweenderivatives of two complementary octamers: CpApTpCpCpGpCpT andApGpCpGpGpApTpG. Each octamer was modified, as shown in the table, sothat hybridization of the corresponding oligodeoxyribonucleotides and ofoligodeoxyribonucleotides having a phosphorothioate backbone wereexamined. The QDN also had the tripeptide based on the residues of1,2dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylic acid (CDPI) (minorgroove binder moiety X) attached either to the 3′ or to the 5′ end (asindicated) through a linking group of the structure L1 or L2. (X, L1 andL2 are the same as in Table 3.) As controls, the melting temperature ofduplexes was also determined for duplexes where the ODNs bore only thelinking groups. As it can be seen in the table, the duplexes aresignificantly stabilized by the presence of a minor groove bindermoiety, and greater stabilization occurs when each strand of the duplexhas a covalently bound minor groove binder.

TABLE 5 T_(m)'s data (° C.) of heterogeneous duplexes carrying 3′- oligo(pyrroloindole carboxamide) peptide residues. Complementary d(AGCGGATG)pd(AICIIATI)p ODNs 3′-L1- 3′-L2-X 3′-L1- 3′-L1-X d(CATCCGCT)p 3′-L1- 4152 11 — 3′-L2-X 57 81 48 67 d(CATCCICT)p 3′-L1- 31 48 −0 41 3′-L1-X 5479 48 63

L1 = —O(CH₂)₆NH— L2 = —OCH₂CH(OH)CH₂NHCOCH₂CH₂NH—

Table 5 discloses melting temperature data obtained when complementaryor “quasi complementary” ODN-MGB were incubated and examined for duplexformation. The minor groove binding moiety X and the linking groups L1and L2 are shown in the table and are the same as in Tables 3 anid 4. Asanticipated, when guanine is replaced inosine (I) in the strands thebinding of the duplexes is very weak (T_(m) is approximately 0° C.) ifthere is no minor groove binding moiety present. However, when guanineis replaced by inosine in the oligonucleotides the presence of onecovalently appended minor groove binder X stabilized the hybrid byalmost 50 ° C. and the presence of two such minor groove binders inantiparallel orientation provided 63° C. of stabilization. When the samestrands contained guanine, one minor groove binder increased the T_(m)by 15° C. while two increased it by nearly 45° C. To the knowledge ofthe present inventors a T_(m) of 81° C. for an 8 mer is unprecedented inthe prior art.

Primer Extension Experiment

That sequence specificity in the Watson-Crick sense of the ODN portionof the ODN-MGB conjugate is required for complexing the ODN-MGBconjugate to a target sequence was demonstrated by a primer extensionexperiment. In this experiment, primer extension occurs with the enzymeT7 DNA polymerase that works from the 5′ end of a template strand. A16-mer ODN-MGB which was complementary in the Watson Crick sense to atarget sequence on the template strand was incubated with a long singlestranded DNA template and the T7 DNA polymerase enzyme. Blockage of theprimer extension was noted at the site of binding with the ODN-MGB whenthe minor groove binding moiety was on the 5′end of the 16-mer ODN. Theminor groove binder was the pyrroloindole tripeptide shown in thisapplication in Table 5. When there was a single mismatch in the sequencespecificity of the 16-mer to the target, primer extension was notblocked. Primer extension was also not blocked when the minor groovebinder moiety was attached to the 3′ end of the 16-mer. Primer extensionwas also not blocked when the sequence specific 16-mer and the freeminor groove binder molecule (Compound 3d, not covalently attached tothe ODN) was incubated with the template and the enzyme. Theseexperiments show that sequence specificity of the ODN-MGB is importantfor complex formation, and that the minor groove binding moiety does notsimply act as an “anchor” to non-specifically bind the appended ODN toanother strand. The ability of ODN-MGB conjugates to inhibit primerextension indicates that these conjugates can be used diagnostically aspolymerase chain reaction (PCR) clamping agents. (see Nucleic AcidResearch (1993) 21: 5332-5336).

Slot-blot Hybridization Assay

The ODN-MGB conjugates of the present invention are useful ashybridization probes. This is demonstrated by the description of thefollowing experiment utilizing a ³²P-labeled ODN-MGB conjugate as adiagnostic probe. When compared to the same ODN without a covalentlylinked minor groove binder (MGB) moiety, the conjugate hybridizes to itscomplement with greater strength, efficiency and specificity. Theslot-blot hybridization assay is a widely used DNA probe diagnosticassay, and the attributes of these MGB-ODN conjugates improve theperformance of the assay.

Specifically, in the herein described experiment a standard protocol wasfollowed, as described in Protocols for Nucleic Acid Blotting andHybridization, 1988, Amersham, United Kingdom. Labelled test ODN whichhybridized to the immobilized plasmid was quantitated as counts perminute (cpm), and plotted vs temperature of hybridization. Four 16-merprobes complementary to M13mp19 DNA (a phage DNA) were evaluated. Two ofthese probes were totally complementary to a site in the phage DNA; oneof these contained a 3′-conjugated CDPI₃ moiety while the other wasunmodified. The second pair of probes were targeted to the same site inM13mp19 DNA but each contained a single mismatch (underlined indrawingFIG. 1). Here again, one ODN was 3′-conjugated to CDPI₃ while the otherwas unmodified.

The results of the slot hybridization study are shown in FIG. 1.Compared to an unmodified but otherwise identical 16-mer, theCDPI₃-containing probe formed a hybrid with a melting temperature(T_(m)) of 50° C. versus only 33° C. This higher melting temperaturemore than doubled the yield of perfectly matched hybrids. When amismatch was introduced into either probe, stability of the respectivehybrids dropped. The CDPI₃-modified probes exhibited good sequencediscrimination between 37-50° C. Furthermore, under the hybridizationconditions used here there was no evidence for binding of the CDPI₃moiety to preexisting double-stranded regions in the M13mp19 DNA target,indicating that totally non-specific binding of these conjugates is notpresent.

Sequence-specific Alkylation of a Gene in Cultured Human Cells

The ODN-MGB conjugates of the present invention which also bear acovalently attached alkylating agent can be used as “anti-gene” agents,that is is for the surpression of the expression of undesired (diseasecausing) genes, provided the ODN-MGB conjugate is complementary to atarget sequence in the target gene.

In such a case the MGB moiety improves the binding to the doublestranded gene (in the presence of a recombinase enzyme) and thealkylating moiety results in permanent covalent binding of the ODN-MGBconjugate to the target sequence.

As a demonstrative experiment the above described 50-mer ODN which was31′ end-modified with a CDPI₃ group and internally modified with anitrogen mustard group (chlorambucil) sequence-specifically crosslinkedto the expected site in a targeted gene (HLA DQβ1 0302 allele) presentin living human BSM cells (a human B-lymphocyte cell line). The ODN-MGBconjugate was added to a suspension of BSM cells at 1-50 μM finalconcentration. After 3.5 hr the genomic DNA was extracted and treatedwith hot pyrrolidine to convert any alkylation events into nicks. Nextthe targeted region of the 0302 allele was amplified by LM-PCR (ligationmediated-polymerase chain reaction), a technique which can be used todetect cleavage events at single-base resolution. Analysis of theproducts on a sequencing gel showed that the modified ODN had bound toand alkylated the targeted site. A similar ODN lacking the CDPI₃ groupwas considerably less effective in efficiency of alkylation of thetarget.

It is probable that in the experiment above the recognition and bindingof the ODN-MGB conjugate to homologous double-stranded DNA took placewith the assistance of nuclear recombinases. In like experiments andapplications endogenous recombinase enzymes can catalyze the sequencespecific targeting of double-stranded genomic DNA by ODN-CDPI₃conjugates in other living cells. When these ODNs have an appendedcrosslinking agent, they can alkylate the targeted DNA. By stabilizingthe D-loop formed in the presence of recombinase, the CDPI₃ promotes thecrosslinkage reaction. The crosslinkage event is a powerful inhibitor ofexpression of the targeted gene. Thus crosslinking ODN-CDPI³ conjugatescan be used as antigene agents.

Specific Embodiments, Experimental Section

General Experimental

All air and water sensitive reactions were carried out under a slightpositive pressure of argon. Anhydrous solvents were obtained fromAldrich (Milwaukee, Wis.). Flash chromatography was performed on 230-400mesh silica gel. Melting points were determined on a Mel-Temp meltingpoint apparatus in open cappilary and are uncorrected. Elementalanalysis was performed by Quantitative Technologies Inc. (Boundbrook,N.J.). UV-visible absorption spectra were recorded.in the 200-400-nmrange on a UV-2100 (Shimadzu) or a Lambda 2 (Perkin Elmer)spectrophotometers. ¹H NMR spectra were run at 20° C. on a Bruker WP-200or on a Varian XL-200 spectrophotometer; chemical shifts are reported inppm downfield from Me₄Si.

2,3,5,6-Tetrafluorophenyl3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate (1a)

2,3,5,6-Tetrafluorophenyl trifluoroacetate (2.6 g, 10 mmol, H. B.Gamper, M. W. Reed, T. Cox, J. S. Virosco, A. D. Adams, A. A. Gall, J.K. Scholler and R. B. Meyer,Jr. Nucleic Acids Res., 1993, Vol. 21, No.1,145-150) was added dropwise to a solution of3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylic acid (1.4g, 6.1 mmol, D. L. Boger, R. S. Coleman, and B. J. Invergo. J.Orq.Chem.,1987, Vol.52, 1521-1530) and triethylamine (1.4 ml, 10 mmol) in 15 ml ofanhydrous DMF. After 1 hr, the reaction mixture was concentrated undervacuum (0.2 mm). The residue was triturated with 2 ml of drydichloromethane. Ethyl ether (50 ml) was added and the mixture was leftat 0° C. overnight. The precipitate was collected by filtration onsintered glass funnel, washed first with 50% ether/CH₂Cl₂ (10 ml), thenwith ether (50 ml) and dried in vacuo. The product was obtained as ayellow solid (1.8 g, 75%): ¹H NMR (Me₂SO-d₆, 200 MHz, ppm) 12.32 (s, 1H,NH), 8.13 (d, 1H, J=9 Hz, C4-H), 8.01 (m, 1H, C₆F₄H), 7.41 (s, 1H,C8-H), 7.26 (d, 1H, J=9 Hz, C5-H), 6.17 (s, 2H, CONH₂), 3.99 (t, 2H, J=9Hz, NCH₂CH₂), 3.30 (t, 2H, J=9 Hz, NCH₂CH₂). Anal. Calcd. forC₁₈H₁₁N₃O₃F₄×2H₂O: C, 50.3; H, 3.52; N, 9.7. Found; C, 50.81; H, 3.60;N, 9.95.

2,3,5,6-Tetrafluorophenyl3-(tert-butyloxycarbonyl)-1,2-dihydro-3H-pyrrolo[3,2e]indole-7-carboxylate(1b)

2,3,5,6-Tetrafluorophenyl trifluoroacetate (2.6 g, 10 mmol) was addeddropwise to a solution of3-(tert-butyloxycarbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylicacid (1.0 g, 3.7 mmol, D. L. Boger, R. S. Coleman, and B. J. Invergo.J.Org.Chem., 1987, Vol.52, 1521-1530) and triethylamine (1.5 ml, 10mmol) in 10 ml of anhydrous CH₂Cl₂. After 4 hrs, CH₂Cl₂ was removed byevaporation at reduced pressure. Flash chromatography (4×20 cm,hexane-ethyl acetate, 1:2) afforded 1b as a yellow crystalline solid(1.25 g, 75%): ¹H NMR (Me₂SO-d₆, 200 MHz, ppm) 12.39 (d, 1H, J=1.4 Hz,NH), 8.02 (m, 1H, C₆F₄H), 7.9 (br s, 1H, C4-H), 7.45 (d, 1H, J=1.4 Hz,C8-H), 7.33 (d, 1H, J=9 Hz, C5-H), 4.02 (t, 2H, J=9 Hz, NCH₂CH₂), 3.26(t, 2H, J=9 Hz, NCH₂CH₂), 1.51 (s, 9H, C(CH₃)₃). Anal. Calcd. forC₂₂H₁₈N₂O₄F₄: C, 58.67; H, 4.03; N, 6.22. Found: C, 58.45; H, 4.09; N,6.13.

3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate DimerMethyl Ester (2a)

A solution of methyl 1,2-dihydro-3H-pyrroloindole-7-carboxylate (0.6 g,1.5 mmol), 1a (0.45 g, 2.25 mmol) and triethylamine (0.2 ml, 1.4 mmol)in 10 ml of anhydrous DMF was incubated at RT for 24 hrs and then at 0°C. for 12 hrs. The resulting insoluble solid was collected byfiltration, washed with DMF (10 ml) and ether (20 ml). Drying in vacuoafforded 2a (0.61 g, 91%) as a pale yellow solid: (¹H NMR (Me₂SO-d₆, 200MHz, ppm) 12.00 (d, 1H, J=1.8 Hz, NH′), 11.54 (s, 1H, NH), 8.28 (d, 1H,J=9 Hz, C4′-H), 7.97 (d, 1H, J=9 Hz, C4-H), 7.33 (d, 1H, J=9 z, C5′-H),7.22 (d, 1H, J=9 z, C5-H), 7.13 (d, 1H, J=1.4 Hz, C8′-H), 6.94 (d, 1H,J=1.1 Hz, C8-H), 6.01 (s, 2H, CONH₂), 4.62 (t, 2H, J=8 Hz, (NH₂CH₂)′),3.98 (t, 2H, J=8 Hz, NCH₂CH₂), 3.88 (s, 3H, CH₃), 3.41 (t, 2H, J=8 Hz,(NCH2CH₂)′), 3.29 (t, 2H, NCH₂CH₂, partially obscured by water). Anal.Calcd. for C₂₄H₂₁N₅O₅×1H₂O×1DMF: C, 58.69; H, 5.84; N, 15.21. Found: C,58.93; H, 5.76; N, 15.82.

3-(tert-Butyloxycarbonyl)-1,2-dihydro-3H-pyrrolo[3.2-e]indole-7-carboxylateDimer Methyl Ester (2c)

A solution of methyl 1,2-dihydro-3H-pyrroloindole-7-carboxylate (0.5 g,2.5 mmol), 1b (1.0 g, 2.2 mmol) and triethylamine (0.1 ml, 0.7 mmol) in10 ml of anhydrous DMF was incubated at RT for 10 hrs and at 0° C. for12 hrs. The resulting insoluble solid was collected by filtration,washed with DMF (5 ml) and ether (40 ml). Drying in vacuo afforded 2c(0.81 g, 74%) as an off-white solid: ¹H NMR (Me₂SO-d₆, 200 MHz, ppm)12.01 (s, 1H, NH′), 11.64 (s, 1H, NH), 8.28 (d, 1H, J=9 Hz, C4′-H), 7.8(br s, 1H, C4-H), 7.32 (apparent t, 2H, C5′-H+C5-H), 7.13 (d, 1H, J=1.1Hz, C8′-H), 6.98 (d, 1H, J=1.1 Hz, C8-H), 4.62 (t, 2H, J=8 Hz,(NH₂CH₂)′), 4.02 (t, 2H, J=8 Hz, NCH₂CH₂), 3.88 (s, 3H, CH₃), 3.41 (t,2H, J=8 Hz, (NCH₂CH₂)′), 3.25 (t, 2H, NCH₂CH₂), 1.52 (s, 9H, C(CH₃)).Anal. Calcd. for C₂₈H₂₈N₄O₅: C, 67.19; H, 5.64; N, 11.19. Found: 66.72,H, 5.69; N, 11.31.

2,3,5,6-Tetrafluorophenyl3-carbamoyl-1,2-dihydro-3H-pyrrolo[3.2-e]indole-7-carboxylate Dimer (2e)

2,3,5,6-Tetrafluorophenyl trifluoroacetate (2.6 g, 10 mmol) was addeddropwise to a suspension of 2b (1.2 g, 2.8 mmol, D. L. Boger, R. S.Coleman, and B. J. Invergo. J.Org.Chem., 1987, Vol.52, 1521-1530) in 15ml of anhydrous DMF. Triethylamine (1.4 ml, 10 mmol) was added and themixture was stirred for 3 hrs. The mixture was concentrated in vacuo(0.2. mm) using rotary evaporator. The residue was triturated with 20 mlof dry dichloromethane. The product obtained was filtered, washed withdichloromethane (10 ml), ether (20 ml), and dried in vacuo to give 2e asa yellow solid (1.5 g, 93%): (¹H NMR (Me₂SO-d₆, 200 MHz, ppm) 12.51 (d,1H, J=1.8 Hz, NH′), 11.58 (s, 1H, NH), 8.39 (d, 1H, J=8.9 Hz, C4′-H),8.04 (m, 1H, C₆F₄H), 7.98 (d, 1H, J=8.8 Hz, C4-H), 7.58 (s, 1H, C8′),7.42 (d, 1H, J=9 Hz, C5′-H), 7.22 (d, 1H, J=9 Hz, C5-H), 6.98 (s, 1H,C8-H), 6.11 (s, 2H, CONH₂), 4.66 (t, 2H, J=7.8 Hz, (NCH₂CH₂)′), 3.94 (t,2H, J=9.1 Hz, NCH₂CH₂), 3.47 (t, 2H, J=8 Hz, (NCH₂CH₂)′), 3.29 (t, 2H,J=9.1 Hz, NCH₂CH₂). Anal. Cacld. for C₂₉H₁₉N₅O₄F₄×1.5H₂O: C, 57.62; H,3.67; N, 11.59. Found: C, 57.18; H, 3.31; N, 11.54.

2,3,5,6-Tetrafluoroihenyl3-(tert-butyloxycarbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylateDimer (2f)

2,3,5,6-Tetrafluorophenyl trifluoroacetate (0.75 g, 2.9 mmol) was addeddropwise to a suspension of 2d (0.25 g, 0.5 mmol, D. L. Boger, R. S.Coleman, and B. J. Invergo. J.Org.Chem., 1987, Vol.52, 1321-1530) andtriethylamine (0.5 ml, 3.5 mmol) in a mixture of anhydrous CH₂Cl₂(8 ml)and DMF (2 ml). The mixture was stirred for 20 hrs. The resulting clearsolution was concentrated in vacuo and was added dropwise to 40 ml of 1Msodium acetate (pH 7.5). The precipitate was centrifuged, washed withwater (2×40 ml), with 10% MeOH in ether(2×40 ml), with ether (40 ml),and with hexane (40 ml). Finally it was dried in vacuo to give 2f as apale yellow solid (0.29 g, 91%): (¹H NMR (Me₂SO-d₆, 200 MHz, ppm) 12.51(s, 1H, NH′), 11.66 (s, 1H, NH), 8.37 (d, 1H, J=8.8 Hz, C4′-H), 8.03 (m,1H, C₆F₄H), 7.8 (br s, 1H, C4-H), 7.58 (s, 1H, C8′-H), 7.40 (d, 1H,J=9.1 Hz, C5′-H), 7.27 (d, 1H, J=8.6 Hz, C5-H), 7.1 (s, 1H, C8-H), 4.65(t, 2H, J=8 Hz, (NCH₂CH₂)′), 4.02 (t, 2H, J=9 Hz, NCH₂CH₂), 3.46 (t, 2H,J=8 Hz, (NCH₂CH₂)′), 3.25 (t, 2H, J=8.9 Hz, NCH₂CH₂), 1.51 (s, 9H,C(CH₃)₃). Anal. Calcd. for C₃₃H₂₆N₄O₅F₄×0.5H₂O: C, 61.59; H, 4.23; N,8.71. Found: C, 61.73; H, 4.12; N, 8.61.

3-carbanoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate TrimerMethyl Ester (3a)

A solution of methyl 1,2-dihydro-3H-pyrroloindole-7-carboxylate (1.0 g,5 mmol), 2e (1.2 g, 2.1 mmol) and triethylamine (0.1 ml, mmol) in 15 mlof anhydrous DMF was incubated at RT for 24 hrs and at 0° C. for 12 hrs.The resulting insoluble solid was collected by filtration, washed withDMF (10 ml), CH₂Cl₂ (20 ml) and ether (20 ml). Drying in vacuo afforded3a (1.1 g, 83%) as a pale yellow solid: (¹H NMR (Me₂SO-d₆, 200 MHz, ppm)12.02 (s, 1H, NH″), 11.75 (s, 1H, NH′), 11.56 (s, 1H, NH), 8.28(apparent t, 2H, J=8.3 Hz, C4-H″+C4′-H), 7.98 (d, 1H, J=9.4 Hz, C4-H),7.98 (d, 1H, J=9 Hz, C4-H), 7.39-7.33 (2 d, 2H, C5″-H+C5′-H), 7.23 (d,1H, J=8.7 Hz, C5-H), 7.14 (d, 1H, J=1.6 Hz, C8″-H), 7.10 (d, 1H, J=1 Hz,C8′-H), 6.97 (s, 1H, C8-H), 6.11 (s, 2H, CONH₂), 4.65 (t, 4H,(NCH₂CH₂)″+(NCH₂CH₂)′), 3.98 (t, 2H, J=8.7 Hz, NCH₂CH₂), 3.88 (S, 3H,CH₃), 3.48-3.25 (m, 6H, (NCH₂CH₂)″+(NCH₂CH₂)′+NCH₂CH₂ partially obscuredwith H₂O). Anal. Calcd. for C₃₅H₂₉N₇O₅×4.5H₂O: C, 59.32; H, 5.0; N,13.03. Found: C, 58.9; N, 5.06; N, 13.77.

2,3,5,6-Tetrafluorophenyl3-carbanoyl-1,2-dihydro-3H-pyrrolo[3,2e]indole-7-carboxylate Trimer (3c)

2,3,5,6-Tetrafluorophenyltrifluoroacetate (2.6 g, 10 mmol) was addeddropwise to a suspension of 3b (1.1 g, 1.8 mmol) in 15 ml of anhydrousDMF and triethylamine (1.4 ml, 10 mmol). The mixture was stirred for 3hrs. The mixture was concentrated in vacuo (0.2 mm). The residue wastriturated with a mixture of dry dichloromethane (20 ml) and methanol (2ml). The resulting product was collected by filtration, washed withdichloromethane (20 ml), ether (20 ml), and dried in vacuo to give 1.3 g(95%) of a yellow-green solid:

(¹H NMR (Me₂SO-d₆, 200 MHz, ppm) 12.54 (d, 1H, J=1 Hz, NH″), 11.79 (s,1H, NH′), 11.56 (S, 1H, NH), 8.41 (d, 1H, J=9.3 Hz, C4-H″), 8.27 (d, 1H,J=9.4 Hz, C4′-H), 8.03 (m, 1H, C₆F₄H), 7.98 (d, 1H, J=9 Hz, C4-H), 7.56(s, 1H, C8″-H), 7.45-7.35 (m, 2H, C5″-H+C5′-H), 7.23 (d, 1H, J=9.2 Hz,C5-H), 7.13 (s, 1H, C8′-H), 6.97 (s, 1H, C8-H), 6.11 (s, 2H, CONH₂),4.65 (m, 4H, (NCH₂CH₂)″+(NCH₂CH₂)′), 3.98 (t, 2H, J=8.7 Hz, NCH₂CH₂),3.45 (m, 4H, (NCH₂CH₂)″+(NCH₂CH₂)′), 3.25 (t, 2H, J=8.7 Hz, NCH₂CH₂).Anal. Calcd. for C₄₀H₂₇N₇O₅F₄×2H₂O: C, 61.59; H, 4.23; N, 8.71. Found:C, 61.73; H, 4.12; N, 8.61.

[3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2e]indole-7-carbox]-1-amido-3-propanolTrimer (3d)

A solution of 3-amino-1-propanol (70 μl, 1.4 mmol), 3c (75 mg,, 0.1mmol) and triethylamine (0.1. ml, mmol) in 2.5 ml of anhydrous DMF wasstirred at RT for 10 hrs. The resultinginsoluble solid was collected byfiltration, washed with DMF (2 ml), CH₂Cl₂ (10 ml) and ether (20 ml).Drying in vacuo afforded 3d (55 mg, 89%) as a pale yellow solid: (¹H NMR(Me₂SO-d₆, 200 MHz, ppm) 11.76 (s, 1H, NH″), 11.65 (s, 1H, NH′), 11.57(s, 1H, NH)), 8.47 (m, 1H, C4-H), 8.24 (m, 1H, C4-H), 7.99 (d, 1H, J=8.4Hz, C4-H), 7.40-7.32 (2d, 2H, C5″-H+C5′-H), 7.23 (d, 1H, J=8.9 Hz,C5-H), 7.12 (s, 1H, C8″-H), 7.10 (s, 1H, C8′-H), 6.99 (s, 1H, C8-H),6.12 (s, 3H, CONH₂+NHCO), 4.66 (t, 4H, (NCH₂CH₂)″+(NCH₂CH₂)′), 3.98 (t,2H, J=8.7 Hz, NCH₂CH₂), 3.51-3.25 (m, 10H,(NCH₂CH₂)″+(NCH₂CH₂)′+NCH₂CH₂+NHCH ₂+CH ₂OH partially obscured withH₂O), 1.70 (p, 2H, J=6.6 Hz, CH₂CH ₂CH₂).

2,3,5,6-Tetrafluorophenyl3-[N-(9-fluorenylmethoxycarbonyl)]aminopropionate (4)

2,3,5,6-Tetrafluorophenyl trifluoroacetate (1.7 g, 6.5 mmol) was addeddropwise to asolution of FMOC-alanine (2.0 g, 6.4 mmol) andtriethylamine (1.0 ml, 7 mmol) in 20 ml of anhydrous CH₂Cl₂. After 1 hr,CH₂Cl₂ was removed by evaporation at reduced pressure using rotaryevaporator, redissolved in 30 ml ethylacetate/hexane (1:1). Flashchromatography (4×20 cm, hexane/ethyl acetate, 3:1) afforded. rude 4 asa white solid. It was recrystallized from hexane/ethyl acetate to give 4as a white crystalline solid (2.3 g, 78%):

¹H NMR (CDCl₃, 200 MHz, ppm) 7.73 (d, 2H, J=7.1 Hz, aromatic protons),7.75 (d, 2H, J=7.7 Hz, aromatic protons), 7.24-7.42 (m, 4H, aromaticprotons), 7.01 (m, 1H, C₆F₄H), 5.21 (br s, 1H, —CONH—), 4.38 (d, 2H,J=7.1 Hz, —CH₂OCO—), 4.20 (m, 1H, benzyl proton), 3.58 (m, 2H, NCH₂),2.93 (t, 2H, J=5.4 Hz, —CH₂CO—). Anal. Calcd. for C₂₄H₁₇NO₄F₄: C, 62.75;H, 3.73; N, 3.05. Found: C, 62.52; H, 3.59: N, 3.01.

1-[3-[N-(9-Fluorenylmethoxycarbonyl)amino]-1-oxopropyl]amino-(R,S)-2,3-propanediol(5)

A solution of 4 (2.0 g, 4.35 mmol) in 20 ml of anhydrous CH₂Cl₂ wasadded to a stirred solution of 3-amino-1,2-propanediol (0.6, mmol) in 10ml MeOH. After 10 min, acetic acid (3 ml) was added and the mixture wasevaporated to dryness. The residue was triturated with 100 ml of water.The obtained solid was filtered off, washed with water and dried byco-evaporation with toluene (2×50 ml) at reduced pressure. Washing with50 ml of ethyl acetate followed by drying in vacuo overnight yielded 5as a white crystalline solid (1.65 g, 99%): ¹H NMR (CDCl₃+MeOD-d4, 200MHz, ppm, Me₄Si): 7.77 (d, 2H, J=7.7 Hz, aromatic protons), 7.61 (d, 2H,J=7.3 Hz, aromatic protons), 7.45-7.29 (m, 4H, aromatic protons), 4.35(d, 2H, J=7.1 Hz, —CH₂OCO—), 4.22 (m, 1H, benzyl proton), 3.72 (m, 1H,—CH— from NHCH₂CHOHCH₂OH), 3.52-3.27 (m, 6H, OCONHCH ₂+CH ₂CHOHCH ₂OH),2.44 (t; 2H, J=6.6 Hz, —CH₂CO—); Anal. Calcd. for C₂₁H₂₄N₂O₅: C, 5.61;H, 6.29; N, 7.29%. Found: C, 65.43; H, 6.28; N, 7.21.

1-[3-[N-(9-Fluorenylmethoxycarbonyl)amino]-1-oxopropyl]amino-(R,S)-2-[[bis(methoxyphenyl)phenylmethoxy]metyl]-2-ethanol(6)

To a stirred solution of 5 (1.6 g, 4.2 mmol) in 30 ml of anhydrouspyridine was added DMTrCl (1.6 g, 4.7 mm ol). After stirring for 3 hrsunder argon, the mixture was evaporated to dryness. Residual pyridinewas removed by co-evaporation with toluene. The residue was dissolved in100 ml of CH₂Cl₂, washed with 2×100 ml water, dried over sodium sulfate,and evaporated to dryness. The residue was purified by flashchromatography (4×20 cm, silica) using ethyl acetate as an eluent. Thefractions containing pure product were combined and evaporated todryness to yield 1.9 g (66%) of 6 as a colorless foam: ¹H NMR (CDCl₃,200 MHz, ppm, Me₄Si) 7.72 (d, 2H, J=7.2 Hz, aromatic protons), 7.56 (d,2H, J=7 Hz, aromatic protons), 7.40-7.20 (m, 13H, aromatic protons),6.80 (d, 4H, J=9 Hz, DMTr protons), 5.76 (br s, 1H, NH), 5.42 (br S, 1H,NH), 4.35 (d, 2H, J=6.6 Hz, —CH₂OCO—), 4.17 (m, 1H, benzyl proton), 3.83(m, 1H, —CH— from NHCH₂CHOHCH₂OH), 3.75 (s, 6H, OCH₃), 3.60-3.30 (m, 4H,OCONHCH ₂+CH ₂CHOHCH₂OH), 3.13 (d, 2H, J=5.4 Hz, CH₂ODMTr), 2.30 (t, 2H,J=5.4 Hz, —CH₂CO—); Anal. Calcd. for C₄₂H₄₂N₂O₇: C, 73.45; H, 6.16; N,4.08. Found: C, 65.43; H, 6.28,N, 7.21.

2,3,5,6-Tetrafluorophenyl1-[3-[N-(9-fluorenylmethoxycarbonyl)amino]-1-oxopronyl]amino-(R,S)-2-[[bis(methoxyphenyl)phenylmethoxy]metyl]-2-ethylButanedioate (7)

To a solution of 6 (1.2 g, 1.75 mmol), triethylamine (0.2 g, 2 moml),1-methylimidazole (20 μl) in 10 ml of anhydrous CH₂Cl₂ was added 0.2 g(2 mmol) of succinic anhydride. This solution was stirred for 20 hrs.Triethylamine (60 μl) was added to the solution followed by 0.6 g (2.2mmol) of 2,3,5,6-tetrafluorophenyl trifluoroacetate. After 1 hr, CH₂Cl₂was removed by evaporation at reduced pressure using a rotaryevaporator, and the residue was dissolved in 15 ml ethylacetate/hexane(1:2). Flash chromatography (4×20 cm, hexane/ethyl acetate, 2:1)afforded 1b as a pale yellow foam (1.2 g, 73%): ¹H NMR (CDCl₃, 300 MHz,ppm, Me₄Si) 7.71 (d, 2H, J=7.2 Hz, aromatic protons), 7.54 (d, 2H, J=7Hz, aromatic protons), 7.40-7.20 (m, 13H, aromatic protons), 7.00 (m,1H, C₆F₄H), 6.78 (d, 4H, J=7 Hz, DMTr protons), 5.71 (br s, 1H, NH),5.42 (br s, 1H, NH), 5.15 (m, 1H, —CH— from NHCH₂CHOHCH₂OH), 4.31 (d,2H, J=6.2 Hz, —CH₂OCO—), 4.16 (d, 5.5 Hz, 1H, benzyl proton), 3.74 (s,6H, OCH₃), 3.60-3.30 (m, 4H, OCONHCH ₂+CH ₂CHOHCH₂OH), 3.20 (br s, 2H,CH₂ODMTr), 2.98 (br s, 2H, COCH₂CH₂CO), 2.72 (br s, 2H, COCH₂CH₂CO),2.20 (br s, 2H, —CH₂CO—); Anal. Calcd. for C₄₂H₄₂N₂O₇: C, 66.80; H,4.96; N, 3.00. Found: C, 66.18; H, 4.98; N, 2.86.

Preparation of CPG Derivative 8

A mixture of 5.0 g of long chain aminoalkyl controled pore glass(LCAA-CPG), 0.5 ml of 1-methylimidazole, and 0.45 g (0.5 mmol) of 7 in20 ml of anhydrous pyridine was swirled in 100 ml flask (orbital mixer,150 rpm). After 3 hrs, the CPG was filtered on a sintered glass funneland washed with 100 ml portions of DMF, acetone, and diethyl ether. TheCPG was dried in vacuo and treated with a mixture of pyridine (20 ml),acetic anhydride (2 ml), and 1-methylimidazole (2 ml). After swirlingfor 30 min, the CPG was washed with pyridine, methanol, and diethylether, then dried in vacuo. The product (8) was analyzed fordimethoxytrityl (DMTr) content according to the literature method (T.Atkinson and M. Smith. in M. Gait (ed.), Oligonucleotide Synthesis. APractical Approach. IRL Press, 1984, Oxford, UK, pp.35-81) and found tohave a loading of 28 μmol/g.

Preparation of CPG Derivative 9

The CPG derivative 8 (3.0 g) was treated twice with 20 ml of 20%piperidine in dry DMF for 5 min each time. The CPG was washed with 100ml portions of DMF, methanol, and diethyl ether, then dried in vacuo.

Preparation of CPG Derivative 10

A mixture of 2.5 g of 9, 7.5 ml of triethylamine, and 0.38 g (0.5 mmol)of 3c in 7.5 ml of anhydrous DMSO was swirled in 50 ml flask (orbitalmixer, 150 funnel rpm). After 2 days, the CPG was filtered on a sinteredglass funnel and washed with 100 ml portions of DMSO, acetone, anddiethyl ether. The CPG was dried in vacuo and treated with a mixture ofpyridine (10 ml), acetic anhydride (1 ml), and 1-methylimidazole (1 ml).After swirling for 30 min, the CPG was washed with DMSO, pyridine,methanol, and diethyl ether, then dried in vacuo.

2-[4-(Phenylazo)benzylthio]ethyl5-[(tert-butyloxy)carboxamido]pentylcarboxylate (11)

6-[(Tert-butyloxy) carboxamido]hexanoic acid (4.16 g, 18 mmol) was driedby co-evaporation with dry DMF (70° C.). The residue was redissolved indry DMF (25 mL) and 2-[4-(phenylazo)-benzylthio]3-ethanol (4.8 g, 15mmol), N,N′-dicyclohexyl carbodiimide (3.71 g, 18 mmol),4-dimethylaminopyridine (1.83 g, 15 mmol) were added at 0° C. Afterstirring at 0° C. for 2 h and 20° C. for 12 h, the reaction mixture wasevaporated to dryness by co-evaporation with butyl acetate, andadditional ethyl acetate (150 mL) was added. This solution was extractedwith 0.7 M HCl (1×30 mL), 5% NaHCO₃ and H₂O (2×50 mL). The organic layerwas dried over Na₂SO₄ and concentrated with rotary evaporator. Washingwith 20 mL of ether and filtration afforded compound 11 (6.91 g, 89%).¹H NMR (CDCl₃, 200 MHz, ppm): 7.91 (m, 4H), 7.52 (m, 5H), 4.48 (t, 2H),4.34 (s, 2H), 3.20 (t, 2H), 3.08 (m, 2H), 2.35 (t, 2H), 1.64-1.2 (m,7H), 1.41 (s, 9H).

1,2,3-benzotriazol-1-yl1-methyl-4-(tert-butyloxy)carboxamido-pyrrole-2-carboxylate

N,N′-Dicyclohexylcarbodiimide (1.1 g, 5.3 mmol) was added to a solutionof 1-methyl-4-[tert-butyloxy)carboxamido]pyrrole-2-carboxylic acid⁴ (1.2g, 5.2 mmol) and 1-hydroxybenzotriazol (0.63 g, 4.7 mmol). Afterstirring for 1 hr, the mixture was filtered through the sintered glassfilter to separate precipitated N,N′-dicyclohexylcarbodiimide. Thefiltrate was evaporated to dryness, redissolved in a mixture of CHCl₃and pentane (1:1), and loaded onto a silica gel column. The fractionscontaining pure product were combined and evaporated to dryness to give1.45 g (80%) of the desired product as a white solid: mp=138-138.5° C.;¹H NMR (CDCl₃, 200 MHz) 8.04 (d, 1H), 7.49-7.40 (m, 4H), 7.09 (d, 1H),3.87 (s, 3H), 1.50 (s, 9H).

2-[4-(Phenylazo)benzylthio]ethyl5-[1-methyl-4-(tert-butyloxy)carboxamido]pyrrole-2-carboxamido]pentylcarboxylate(12)

Trifluoroacetic acid (5 mL) was added at 0° C. to 11 (0.73 g, 1.5 mmol).After stirring at0° C. for 20 min the reaction mixture was evaporated todryness by co-evaporation with CHCl₃. The residue was dissolved in dryCH₂Cl₂ (15 mL) and 1,2,3-benzotriazol-1-yl1-methyl-4-(tert-butyloxy)carboxamido-pyrrole-2-carboxylate (0.59 g,1.65 mmol), dry triethylamine (0.23 g, 2.3 mmol) were added. Afterstirring at ambient temperature for 15 min, CHCl₃ was added (100 mL).The reaction mixture was extracted with 5% NaHCO₃ (2×20 mL), H₂O (2×20mL). The organic layer was dried over Na₂SO₄ and concentrated on arotary evaporator. Chromatography on silica gel (100 g) with CHCl₃afforded 0.88 g (91.8%) 12. ¹H NMR (CDCl₃, 200 MHz, ppm): 7.88 (m, 4H),7.46 (m, 5H), 6.74 (s, 1H), 6.38 (s, 1H), 6.26 (s, 1H ), 5.87 (t, 1H),4.18 (t, 2H, J=6 Hz), 3.82 (s, 3H), 3.79 (s, 2H), 3.3 (m, 2H), 2.63 (t,2H, J=6 Hz), 2.30 (t, 2H, J=6 Hz), 1.64-1.2 (m, 6H), 1.46 (s, 9H).

2-[4-(Plenylazo)benzylthio]ethyl5-[1-methyl-4-[1-methyl-4-(tert-butyloxy)carboxamidopyrrole-2-carboxamido]pyrrole-2-carboxamido]pentylcarboxylate(13)

A solution of 12 (2.43 g, 4 mmol) in dry CH₂Cl₂ (8 mL) was treated withtrifluoroacetic acid (4 mL) at 0° C. The resulting solution was left atambient temperature in stopped flask for 1 h and then partitionedbetween 30% aqueous K₂CO₃ (30 mL) and CH₂Cl₂ (30 mL). The lower layerwas collected. The aqueous phase was extracted with dichloromethane(2×20 mL), and the combined organic extracts, after being washed withH₂O (1×20 mL), were dried over Na₂SO₄ and evaporated. The residue wasdissolved in CH₂Cl₂ (10 mL) and 1,2,3-benzotriazol-1-yl1-methyl-4-(tert-butyloxy) carboxamidopyrrole-2-carboxylate (1.43 g, 4mmol) and dry triethylamine. (0.8 g, 8 mmol) were added. After. stirringat ambient temperature for 30 min, CHCl₃ (100 mL) was added. Thereaction mixture was extracted with 5% NaHCO₃ (2×20 mL), H₂O (2×20 mL).The organic layer was dried over Na₂SO₄ and concentrated on a rotaryevaporator. Chromatography on silica gel (100 g) with CHCl₃ afforded1.95 g (66.8%) of 13. ¹H NMR (CDCl₃, 200 MHz, ppm): 7.87 (m, 4H), 7.46(m, 5H), 7.04 (d, 1H, J=1.5 Hz), 6.77 (br s, 1H), 6.52 (br s, 1H), 6.50(d, 1H, J=1.5 Hz), 6.31 (br s, 1H), 5.95 (t, 1H), 4.19 (t, 2H, J=6 Hz),3.85 (s, 6H), 3.78 (s, 2H), 3.32 (m, 2H), 2.64 (t, 2H, J=6 Hz), 2.31 (t,2H, J=6 Hz), 1.64-1.2 (m, 6H), 1.48 (s, 9H).

2-[4-(Phenylazo)benzylthio]ethyl5-[1-methyl-4-[1-methyl-4-[1-methyl-4-(tert-butyloxy)carboxamidopyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pentylcarboxylate(14)

A solution of 13 (1.90 g, 2.6 mmol) in dry CH₂Cl₂ (6 mL) was treatedwith trifluoroacetic acid (3 mL) at 0° C. The resulting solution wasleft at ambient temperature in stopped flask for 1 h and thenpartitioned between 30% aqueous K₂CO₃ (30 mL) and CH₂Cl₂ (30 mL). Thelower layer was collected. The aqueous phase was extracted withdichloromethane (2×20 mL), and the combined organic extracts, afterbeing washed with H₂O (1×20 mL), were dried over Na₂SO₄ and evaporated.The residue was dissolved in CH₂Cl₂ (2.5 mL) and 1,2,3-benzotriazol-1-yl1-methyl-4-(tert-butyloxy)carboxamidopyrrole-2-carboxylate (1.4 g, 3.9mmol), dry triethylamine (0.8 g, 8 mmol) were added. After stirring atambient temperature for 1 h, CHCl₃ (100 mL) was added. The reactionmixture was extracted with 5% NaHCO₃ (2×20 mL), H₂O (2×20 mL). Theorganic layer was dried over Na₂SO₄ and concentrated on arotary.evaporator. Chromatography on silica gel (100 g) with 0-1.5%methanol in CHCl₃ afforded 1.56 g (70.5%) of 14. ¹H NMR (CDCl₃, 200 MHz,ppm): 7.87 (m, 4H), 7.68 (br s, 1H), 7.60 (br s, 1H), 7.46 (m, 5H), 7.08(br s, 2H), 6.78 (br s, 1H), 6.56 (d, 1H, J=1.5 Hz), 6.60 (br s, 1H),6.55 (d, 1H, J=1.5 Hz), 6.03 (t, 1H), 4.18 (t, 2H, J=6 Hz), 3.86 (m,9H), 3.78 (s, 2H), 3.32 (m, 2H), 2.63 (t, 2H, J=6 Hz), 2.30 (t, 2H, J=6Hz), 1.64-1.2 (m, 6H), 1.48 (s, 9H).

2-[4-(Phenylazo)benzylthio]ethyl-5-[1-methyl-4-[1-methyl-4-[1-methyl-4-[1-methyl-4-(tert-butyloxy)carboxamidopyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pentylcarboxylate(15)

A solution of 14 (0.32 g, 0.32 mmol) in dry CH₂Cl₂ (5 mL) was treatedwith trifluoroacetic acid (2.5 mL) at 0° C. The resulting solution wasleft at ambient temperature in stopped flask for 1 h and thenpartitioned between 30% aqueous K₂CO₃ (30 mL) and CH₂Cl₂ (30 mL). Thelower layer was collected. The aqueous phase was extracted withdichloromethane (2×20 mL), and the combined organic extracts, afterbeing washed with H₂O (1×20 mL), were dried over Na₂SO₄ and evaporated.The residue was dissolved in CH₂Cl₂ (1 mL) and 1,2,3-benzotriazol-1-yl1-methyl-4-(tert-butyloxy)carboxamidopyrrole-2-carboxylate (0.11 g, 0.32mmol), dry triethylamine (0.06 g, 0.03 mmol) were added. After stirringat ambient temperature for 1.5 h, CHCl₃ (100 mL) was added. Thesuspension was filtered and the filtrate was extracted with 5% NaHCO₃(2×20 mL), H₂O (2×20 mL). The organic layer was dried over Na₂SO₄ andevaporated to dryness. The yield of 15 was 0.25 g (80%). ¹H NMR (CDCl₃,200 MHz, ppm): 8.17 (br s, 1H), 7.98 (br s,), 7.96 (br s,), 1H 7.85 (m,4H), 7.44 (m, 5H), 7.09 (br s, 2H), 7.02 (s, 1H), 6.78 (br s, 1H), 6.74(br s, 1H), 6.66 (s, 1H), 6.58 (s, 3H), 6.29 (t, 1H), 4.18 (t, 2H, J=6Hz), 3.78 (m, 14H), 3.28 (m, 2H), 2.60 (t, 2H, J=6 Hz), 2.26 (t, 2H, J=6Hz), 1.64-1.2 (m, 6H), 1.48 (s, 9H).

2-[4-(Phenylazo)benzylthio]ethyl5-[1-methyl-4-[1-methyl-4-[1-methyl-4-[1-methyl-4-[1-methyl-4-(tert-butyloxy)carboxamidopyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pentylcarboxylate(16)

A solution of 15 (0.65 g, 0.67 mmol) in dry CH₂Cl₂ (10 mL) was treatedwith trifluoroacetic acid (5 mL) at 0° C. The resulting yellowishsolution was left at ambient temperature in stopped flask for 1 h andthen partitioned between 30% aqueous K₂CO₃ (30 mL) and CH₂Cl₂ (30 mL).The lower layer was collected. The aqueous phase was extracted withdichloromethane (2×20 mL), and the combined organic extracts, afterbeing washed with H₂O (1×20 mL), were dried over Na₂SO₄ and evaporated.The residue was dissolved in DMF (1 mL) and 1,2,3-benzotriazol-1-yl1-methyl-4-(tert-butyloxy)carboxamidopyrrole-2-carboxylate (0.24 g, 0.67mmol), dry triethylamine (0.13 g, 0.67 mmol) were added. After stirringat ancient temperature for 3 h, the reaction mixture was evaporated todryness by co-evaporation with butyl acetate. The residue was dissolvedin 3 mL 2.5% DMF in CHCL₃. Chromatography on silica gel (100 g) with0-2.5% methanol in CHCl₃ (2.5% DMF) afforded 0.67 g (45%) of 16.

2,3,5,6-Tetrafluorophenyl-4′-[bis(2-chloroethyl)amino]phenylbutyrate(Chlorambucil 2,3,5,6-tetrafluorophenyl Ester)

To a solution of 0.25 g (0.82 mmol) of chlorambucil (supplied by FlukaA. G.), 0.3 g (1.1 mmol) of 2,3,5,6-tetrafluorophenyl trifluoroacetatein 5 ml of dry dichloromethane was added 0.2 Ml of dry triethylamine.The mixture was stirred under argon at room temperature for 0.5 h andevaporated. The residual oil was purified by column chromatography onsilica gel with hexane-chloroform (2:1) as the eluting solvent to givethe ester as an oil: 0.28 g (75%); TLC on silica gel (CHCl₃) R_(f) 0.6;IR (in CHCl₃) 3010, 1780, 1613, 1521, 1485 cm⁻¹.

Introduction of Chlorambucil Residue into the Primary Amino Groups ofOligonucleotides

Preparation of the cetyltrimethylammonium salt of oligonucleotides: a100 μL of aqueous solution of oligonucleotide (50-500 ug), generallytriethylammonium salt, was injected to a column packed with Dowex 50wx8in the cetyltrimethylammonium form and prewashed with 50% alcohol inwater. The column was eluted by 50% aqueous ethanol (0.1 mL/min).Oligonucleotide containing fraction was dried on a Speedvac over 2 hoursand used in following reactions.

Ethanol solution (50 uL) of cetyltrimethylammonium salt of anoligonucleotide (50-100 μg) was mixed with of 0.08 M solution of2,3,5,6-tetrafluorophenyl-4′-[bis(2-chloroethyl)amino]phenylbutyrate(tetrafluoro-phenyl ester of chlorambucil) in acetonitrile (50 μL) and 3gL of diisopropylethylamine. After shaking for three hours at roomtemperature, the product was precipitated by 2% LiClO₄ in acetone (1.5mL). The product was reprecipitated from water (60 uL) by 2% LiClO₄ inacetone three times. Finally chlorambucil derivative of oligonucleotidewas purified by Reverse Phase Chromatography with approximately 50-80%yield. The fraction containing a product was concentrated byapproximately butanol. Isolated chlorambucil derivative ofoligonucleotide was precipitated in acetone solution of LiClO₄, washedby acetone and dried under vacuum of oil pump. All manipulation ofreactive oligonucleotide was performed as quickly as possible, with theproduct in ice-cold solution, starting from the chromatographic fractioncollected.

Oligonucleotide Synthesis

All oligonucleotides were prepared from 1 μmol of the appropriate CPGsupport on an ABM 394 using protocol supplied by manufacturer. Standardreagents for the -cyanoethylphosphoramidite coupling chemistry werepurchased from Glen Research. 5′-aminohexyl modifications wereintroduced using an N-MMT-hexanolamine phosohoramidite linker (GlenResearch). 3′-aminohexyl modifications were introduced using the CPGprepared as previously described, C. R. Petrie, M. W. Reed, A. D. Adams,and R. B. Meyer, Jr. Bioconjuqate Chemistry, 1992, 3, 85-87.

Preparation of Coniucates (Reaction Scheme 3)

To a solution of cetyltrimethylammonium salt of an aminohexyl modifiedoligonucleotide (30-50 nmol, Jost, J.-P., Jiricny, J., and Saluz, H.(1989) Quantitative precipitation of short oligonucleotides with lowconcentratoins of cetyltrimethylammonium bromide. Nucleic Acids Res. 17,2143) and 1.5 μl of N,N-diisopropylethylamine in 40 μl of dry DMSO wasadded 40 μl of 4 mM solution of the TFP ester (1a, 1b, 2e, 2f or 3c).The reaction mixture was kept for 12 hrs at RT. The oligonucleotiderelated material was precipitated by addition of1.5 ml of 2% LiClO₄ inacetone. The pellet was washed with acetone, and dried in vacuo. Thepellet was redissolved in 60 μl of 50% DMF in H₂O and precipitated againas described above using 2% solution of LiClo₄ in acetone. Thisprocedure was repeated twice. The residue was purified by HPLC (4.6×250mm, C-18, Dynamax-300A, Rainin) using a gradient of acetonitrile from 20to 75% in the presence of 50 mM LiClO₄. The fraction containing pureproduct was dried in vacuo using speedvac. The residue was dissolved in60-80 μl of H₂O and precipitated with 1.5 ml of 2% LiClO₄ in acetone.After washing with acetone (2×1.5 ml) and drying in vacuo, the pelletwas dissolved in 100 μl of H₂O. The yield of final product was 20-50%.

A modified procedure of Godovikova et al. (T. S. Godovikova, V. F.Zarytova, T. V. Maltzeva, L. M. Khalim-skaya. Bioorgan. Khim., 1989, 15,1246-1259) was used for the preparation of the oligonucleotideconjugates bearing 4-amino-N-methylpyrrol-2-carboxylic acid residues. Asolution of cetyltrimethylammonium salt of 3′-phosphate-containingoligonucleotide (50-100 nmol), triphenylphospine (10 mg),2,2′-dipyridyldisulfide (10 mg), N,N-dimethylaminopyridine (10 mg), andone of the analogues selected from compounds 11 through 16 in 100 μl ofdry DMF was incubated for 20 min at RT. The oligonucleotide relatedmaterial was precipitated by addition of 1.5 ml of 2% LiClO₄ in acetone.The pellet was washed with acetone, and dried in vacuo. The residue waspurified by HPLC using gradient of acetonitrile from 20 to 75% inpresence of 50 mM LiClO₄. The fraction containing pure product was driedin vacuo using speedvac. The residue was dissolved in 60-80 μl of H₂Oand precipitated with 1.5 ml of 2% LiClO₄ in acetone. Afterwashing withacetone (2×1.5 ml) and drying in vacuo, the pellet was dissolved in 100μl of H₂O. The yield of final product was 30-50%.

Preparation of Coniuqates (Reaction Scheme 4)

CPG containing 5′-aminohexyl derivatized oligonucleotide obtained in asynthesis on 1 μmol scale was treated with 2% dichloroacetic acid inCH₂Cl₂ to remove the 9-fluorenylmethoxycarbonyl (Fmoc) protecting groupfrom the amino group followed by washing with acetonitrile, and dryingby flushing with argon. The CPG was transferred into 1.5 ml plastic tubeand 100 ul of 50 mM solution of TFP ester in anhydrous DMSO was added.The tube was shaken for 24 hrs, then washed with 3×1.5 ml DMSO, 2×1.5 mlacetone, and dried in vacuo. The CPG was treated with concentratedammonia to deprotect oligonucleotide using standard conditions. Theresulting reaction mixture was separated using reverse phase HPLC asdescribed above. Typical yield was about 50%.

Thermal Denaturation Studies

Optical melting curves of oligonucleotide complexes bearing4-amino-N-methylpyrrol-2-carboxylic acid residues were obtained in 200mM NaCl, 10 mM Na₂HPO₄, 0.1 mM EDTA (pH 7.0) on the UV detector of aMilichrom liquid chromatograph in a thermoregulated cell speciallydesigned for this purpose. The data were collected and processed on apersonal computer as described by S. G. Lokhov et al. (S. G. Lokhov, M.A. Podyminogin, D. S. Sergeev, V. N. Silnikov, I. V. Kutyavin, G. V.Shishkin, V. F. Zarytova. Bioconjugate Chem. 1992, 3, 414).

The oligonucleotide complexes carrying1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylic acid (CDPI) residueswere melted in 140 mM KCl, 10 mM MgCl₂, 20 mM HEPES-HCl (pH 7.2) on aLambda 2 (Perkin Elmer) spectrophotometer with a PTP-6 automaticmulticell temperature programmer. The melting temperatures of complexes(Tm) were determined from the derivative maxima.

Primer Extension Reactions

Primer extension reactions were performed as previously described byLee, et al., [Biochemistry (1994) 33: 6024-6030]. The finalconcentrations of template, primer and blocking ODNs were 5×10⁻¹⁰ M,4×10⁻⁸ M and 10⁻⁹ M, respectively. Primer extension was carried out for15 min at 45° C., and the products were analyzed by denaturing gelelectrophoresis as described in the reference.

In the absence of any blocking ODN, the primer extension reactiongenerated a high molecular weight product which ran as an unresolvedband in the sequencing gel. Weak bands corresponding to pause sites orto spontaneous termination events were reproducibly observed in allreaction mixtures. Unmodified 16-mer and 32-mer ODNs, fullycomplementary to the target, failed to block primer extension. Alsowithout activity were complementary 8-mer and 16-mer ODNs, each of whichwas 3′-linked to a CDPI₃ group. Only a fully complementary 16-mer ODNwith a 5′-conjugated CDPI₃ group arrested primer extension by T7 DNApolymerase. A complementary 8-mer ODN with the same 5′ modificationgenerated only a trace amount of blocked product. Control ODNs confirmedthat inhibition of primer extension required both a complementary ODNand a covalently linked MGB. Two singly-mismatched 16-mer ODNs, eachwith a 5′-linked CDPI₃ peptide, were much less inhibitory than theperfectly matched ODN-MGB conjugates. Addition of unmodified 16-mer ODNtogether with an equimolar amount of free CDPI₃ had no effect on primerextension, emphasizing the importance of the conjugation of the MGB tothe ODN. When a 5′ acridine moiety was conjugated to the fullycomplementary 16-mer ODN instead of the MGB, a loss of inhibitoryactivity was seen.

Cell Culture Crosslinking Experiment

The ODN-MGB conjugate was complementary to nucleotides 815-864 of thetemplate strand of the DQβ1 allele [Proc. Natl. Acad. Sci. USA (1983)80: 7313-7317]. The human BSM B-cells used here are homozygous for thisallele and express it constitutively. Prior to adding the ODN, the BSMcells were grown in a 25 ml flask to a density of 4.5×10⁶ cells per mlof media. For each treatment the cells from a 2 ml aliquot of culturewere pelleted and resuspended in 200 ul of serum free media whichcontained 0, 1, 10 or 50 μM 50-mer chlorambucil-linked ODN (either withor without a 3′ conjugated CDPI₃ group). Each sample was incubated for3.5 hours at 37° C. with 5% CO₂ in a 48-well microtiter plate. The cellswere then transferred to Eppendorf 0.5 ml centrifuge tubes, pelleted 5min at 2,000 rpm, washed twice with 500 μl phosphate buffered saline(PBS) and deproteinized with Proteinase K/SDS overnight at 370° C. Afterphenol/chloroform extraction and Rnase A digestion the DNA was treatedwith 1M pyrrolidine at 90° C. for 30 mn. Pyrrolidine was removed byethanol precipitation, and the ligation-mediated ploymerase chainreaction (PCR) reaction was performed as described by Lee et al.[Biochemistry (1994) 33: 6024-6030]. Amplified DNA was analyzed on asequencing gel to visualize any sequence specific nicking that mighthave resulted from alkylation of the target by thechlorambucil-containing ODNs. Results showed cleavage at the nucleotideon the target adjacent to the crosslinker on the ODN, and that theCDPI₃-containing 50-mer was 10-fold more efficient than the same ODNwithout the MGB in sequence specifically alkylating the 0302 allele.Complete media was prepared from the following components (the serumfree media lacked HI-FCS):

500 ml RPMI 1640 with L-Glutamine (2 mM) (Gibco BRL Cat. No. 11875-036)

50 ml of HI-FCS (Gibco BRL Cat. No. 26140, heat inactivated 30 min at55° C.)

5 ml of 100×Penn/Strep (Gibco BRL Cat. No. 15070-022)

5 ml of 200 mM L-Glutamine (Gibco BRL Cat. No. 25030-024)

5 ml of 100×Sodium Pyruvate (11 mg/ml; made from Gibco BRL Cat. No.11840-030)

5 ml of 1 M HEPES, pH 7.3 (Gibco BRL Cat. No. 15630-023)

12 50 base pairs nucleic acid single linear DNA modified_base 26/mod_base= OTHER /note= “N = 5-(3-aminopropyl)-2′-deoxyuridine withchlorambucil attached to the amino group” modified_base 50 /mod_base=OTHER /note= “N = adenosine modified by3-carbamoyl-1,2-dihydro-3H-pyrrolo[3, 2e]indole-7-carboxylate trimer(CDPI-3)” 1 GGTTATTTTT GAAGATACGA ATTTCNCCAG AGACACAGCA GGATTTGTCN 50 16base pairs nucleic acid single linear DNA 2 TTTTTTTTTT TTTTTT 16 16 basepairs nucleic acid single linear DNA modified_base 16 /mod_base= OTHER/note= “N = thymidine modified by 6-aminohexanoic acid(-NH(CH-2)-6COOH)” 3 TTTTTTTTTT TTTTTN 16 16 base pairs nucleic acidsingle linear DNA modified_base 16 /mod_base= OTHER /note= “N =thymidine modified by minor groove binder moiety represented by X, wherem = one 4-amino-N-methylpyrrol-2-carboxylic acid residue” 4 TTTTTTTTTTTTTTTN 16 16 base pairs nucleic acid single linear DNA modified_base 16/mod_base= OTHER /note= “N = thymidine modified by minor groove bindermoiety represented by X, where m = two4-amino-N-methylpyrrol-2-carboxylic acid residues” 5 TTTTTTTTTT TTTTTN16 16 base pairs nucleic acid single linear DNA modified_base 16/mod_base= OTHER /note= “N = thymidine modified by minor groove bindermoiety represented by X, where m = three4-amino-N-methylpyrrol-2-carboxylic acid residues” 6 TTTTTTTTTT TTTTTN16 16 base pairs nucleic acid single linear DNA modified_base 16/mod_base= OTHER /note= “N = thymidine modified by minor groove bindermoiety represented by X, where m = four4-amino-N-methylpyrrol-2-carboxylic acid residues” 7 TTTTTTTTTT TTTTTN16 16 base pairs nucleic acid single linear DNA modified_base 16/mod_base= OTHER /note= “N = thymidine modified by minor groove bindermoiety represented by X, where m = five4-amino-N-methylpyrrol-2-carboxylic acid residues” 8 TTTTTTTTTT TTTTTN16 16 base pairs nucleic acid single linear DNA 9 CCAGCAGAAG ATAAAA 1616 base pairs nucleic acid single linear DNA modified_base 16 /mod_base=OTHER /note= “N = adenosine modified by3-carbamoyl-1,2-dihydro-3H-pyrrolo[3, 2e]indole-7-carboxylate trimer(CDPI-3)” 10 CCAGCAGAAG ATAAAN 16 16 base pairs nucleic acid singlelinear DNA 11 CCAGCAGAAG ATCAAA 16 16 base pairs nucleic acid singlelinear DNA modified_base 16 /mod_base= OTHER /note= “N = adenosinemodified by 3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2e]indole-7-carboxylate trimer (CDPI-3)” 12 CCAGCAGAAG ATCAAN 16

What is claimed is:
 1. An oligonucleotide-minor groove binder-reportergroup combination comprising an oligonucleotide having a plurality ofnucleotide units, a 3′-end and a 5′-end, a minor groove binder moietyattached to at least one of said nucleotide units through a linkinggroup which covalently binds the minor groove binder moiety to theoligonucleotide, and a reporter group; wherein the minor groove bindermoiety is a radical of a molecule having a molecular weight ofapproximately 150 to approximately 2000 Daltons that binds in anon-intercalating manner into the minor groove of double stranded DNA,RNA or hybrids thereof with an association constant greater thanapproximately 10³ M⁻¹.
 2. An oligonucleotide-minor groovebinder-reporter group combination of claim 1 wherein the minor groovebinding moiety is attached to the 5′ -end of the oligonucleotide.
 3. Anoligonucleotide-minor groove binder-reporter group combination of claim1 wherein the minor groove binding moiety is attached to the 3′-end ofthe oligonucleotide.
 4. An oligonucleotide-minor groove binder-reportergroup combination of claim 1 wherein the minor groove binder moiety isattached to a nucleotide unit which is neither at the 3′ nor at the 5′end of the oligonucleotide.
 5. An oligonucleotide-minor groovebinder-reporter group combination of claim 1 wherein the minor groovebinder moiety is attached to the heterocyclic base portion of anucleotide unit.
 6. An oligonucleotide-minor groove binder-reportergroup combination of claim 1, wherein the reporter group is covalentlyattached to the minor groove binder moiety.
 7. An oligonucleotide-minorgroove binder-reporter group combination of claim 1, wherein saidreporter group is detectable by color, ultraviolet spectrum, or aphysical or chemical characteristic.
 8. An oligonucleotide-minor groovebinder-reporter group combination of claim 1, wherein said reportergroup is a diazobenzene moiety.
 9. An oligonucleotide-minor groovebinder-reporter group combination of claim 1, wherein said reportergroup is attached to said minor groove binder moiety viaa—NH(CH₂)_(m)COO(CH₂)_(m)S(CH₂)_(m)—bridge wherein each subscript m isan integer between 1 and
 10. 10. An oligonucleotide-minor groovebinder-reporter group combination of claim 1, wherein said minor groovebinder moiety is represented by the formula:

wherein the subscript m is an integer of from 2 to 5; and R is aselected from the group consisting of tBOC and CONH₂.
 11. Anoligonucleotide-minor groove binder-reporter group combination of claim1, wherein said oligonucleotide is an oligodeoxyribonucleotide.
 12. Anoligonucleotide-minor groove binder-reporter group combination of claim1, wherein said oligonucleotide is an oligodeoxyribonucleotide havingnucleotide units interconnected by phosphodiester linkages,phosphorothioate linkages, methylphosphonate linkages or combinationsthereof.
 13. An oligonucleotide-minor groove binder-reporter groupcombination of claim 1, wherein said oligonucleotide comprises basesselected from the group consisting of uracil, guanine, adenine,cytosine, thymine, hypoxanthine, 2-aminoadenine, 2-thiouracil,2-thiothymine, 5-N⁴ ethenocytosine, 4-aminopyrazolo[3,4-d]pyrimidine and6-amino-4-hydroxy-[3,4-d]pyrimidine.